MHC class II-peptide conjugates useful in ameliorating autoimmunity

ABSTRACT

The present invention is directed to complexes consisting essentially of an isolated MHC component and an autoantigenic peptide associated with the antigen binding site of the MHC component. These complexes are useful in treating autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/690,840, now U.S. Pat. No. 5,260,422, which is acontinuation-in-part of U.S. application Ser. No. 07/576,084, now U.S.Pat. No. 5,130,297, which is a continuation of U.S. application Ser. No.07/210,594, filed Jun. 23, 1988, now abandoned. It is also acontinuation-in-part of U.S. application Ser. No. 07/635,840, filed Dec.28, 1990, now U.S. Pat. No. 5,284,935, which is a continuation-in-partof U.S. application Ser. No. 07/367,750 filed Jun. 21, 1989, now U.S.Pat. No. 5,194,425, all of which are, in their entirety, incorporatedherein by reference.

BACKGROUND OF THE INVENTION TECHNICAL FIELD

The invention relates to the methods and compositions for the modulationof T cell function in the treatment of for example, autoimmune diseases,allergic responses, transplant rejection, and other immunologicaldisorders. In particular, it concerns complexes which target helper Tcells by using a complex of the major histocompatibility complex (MHC)glycoproteins with peptides representing fragments of antigensassociated with such diseases. These complexes can be further conjugatedto radioisotopes or other labels for diagnostic purposes, or to toxinsor other substances which render the complexes therapeutically useful.

BACKGROUND OF THE INVENTION

A number of pathological responses involving unwanted T cell activationare known. For instance, a number of allergic diseases, have beenassociated with particular MHC alleles or suspected of having anautoimmune component.

Other deleterious T cell-mediated responses include the destruction offoreign cells that are purposely introduced into the body as grafts ortransplants from allogeneic hosts. This process, known as "allograftrejection," involves the interaction of host T cells with foreign MHCmolecules. Quite often, a broad range of MHC alleles are involved in theresponse of the host to an allograft.

Autoimmune disease is a particularly important class of deleteriousimmune response. In autoimmune diseases, self-tolerance is lost and theimmune system attacks "self" tissue as if it were a foreign target. Morethan 30 autoimmune diseases are presently known; these include manywhich have received much public attention, including myasthenia gravis(MG) and multiple sclerosis (MS).

A crude approach to treating autoimmune disease and otherimmunopathologies is general immunosuppression. This has the obviousdisadvantage of crippling the ability of the subject to respond to realforeign materials to which it needs to mount an immune response. An onlyslightly more sophisticated approach relies on the removal of antibodiesor immune complexes involving the target tissue. This also has adverseside effects, and is difficult to accomplish. The invention approach,described in detail below, relies on a "clonotypic" reagent--i.e., areagent which attacks only the cells of the immune system which areresponsive to the autoantigen.

In the general paradigm now considered to describe the immune response,specific antigens presented result in a clonal expansion, as firstproposed by Burnet in 1959. According to this scenario, a particularsubject will have hundreds of thousands of T and B cells each bearingreceptors that bind to different antigenic determinants. Upon exposureto an antigen, the antigen selectively binds to cells bearing theappropriate receptors for the antigenic determinants it contains,ignoring the others. The binding results in a cloned population ofthousands of daughter cells, each of which is marked by the samereceptor. A clonotypic reagent affects only a subset of the T and Bcells which are appropriate for the antigen of interest. In the case ofthe invention compositions, the antigenic determinant is usually thatassociated with an autoimmune disease.

The clonotypic reagent compositions of the invention are specificallydesigned to target T-helper cells which represent the clones specificfor the antigenic determinant(s) of the tissue which is affected by theautoimmune disease. T-helper cells recognize a determinant only inassociation with an MHC protein; the complexes of the inventiontherefore include an effective portion of the MHC protein.

There have, recently, been some related approaches which attempt tointerdict the immune response to specific antigens. For example, theautoantigen thyroglobulin has been conjugated to ricin A and theconjugate was shown to suppress specifically the in vitro antibodyresponse of lymphocytes which normally respond to this antigen. It wassuggested that such immunotoxins would specifically deleteautoantibody-secreting lymphocyte clones (Rennie, D. P., et al., Lancet(Dec. 10, 1983) 1338-1339). Diener, E., et.al., Science (1986)233:148-150 suggested the construction of compounds which causeantigen-specific suppression of lymphocyte function by conjugatingdaunomycin to the hapten (in this case, of ovalbumin) using anacid-sensitive spacer. The conjugate caused hapten-specific inhibitionof antibody secretion by B lymphocytes in vitro and in vivo. A conjugateof daunomycin (with an acid-sensitive spacer) to a monoclonalantibody-specific to T cells also eliminated the response byT-lymphocytes to concanavalin A. Steerz, R. K. M., et al., J. Immunol.(1985) 134:841-846 utilized radiation as the toxic element in a toxinconjugate. Rats were administered a radioactively labeled, purifiedreceptor from electric fish, prior to injection with cold receptor.Injection with this receptor is a standard procedure to induceexperimental autoimmune myasthenia gravis (EAMG). Control rats thatreceived preinjection only either of cold receptor or radiolabeledalbumin, prior to administration of receptor to induce the diseasedevelop the symptoms of EAMG; those pretreated withradioactively-labeled receptor showed reduced symptoms. It was surmisedthat the labeled, and therefore destructive, receptor selectivelyeliminated immunocompetent cells. Similar work utilizing aricin/receptor conjugate for pretreatment was reported by Killen, J. A.,et al., J. Immunol. (1984) 133:2549-2553.

A less specific approach which results in the destruction of T cells ingeneral is treatment with an IL-2/toxin conjugate as reported by Hixson,J. R., Medical Tribune (Jan. 28, 1988) 4-5. In a converse, but related,approach Liu, M. A., et al., Science (1988) 239:395-397, report a methodto "link up" cytotoxic T cells with a desired target, regardless of thecytotoxic T cell specificity. In this approach, antibody specific to theuniversal cytotoxic T-lymphocytes to destroy human melanoma cells whenmelanocyte-stimulating hormone was the hormone used.

The current model of immunity postulates that antigens mobilize animmune response, at least in part, by being ingested by anantigen-presenting cell (APC) which contains on its surface a Class IIglycoprotein encoded by a gene in the major histocompatibility complex(MHC). The antigen is then presented to a specific T helper cell in thecontext of the surface bound MHC glycoprotein, and by interaction of theantigen specific T cell receptor with the antigen-MHC complex, the Thelper cell is stimulated to mediate the antigen-specific immuneresponse, including induction of cytotoxic T cell function, induction ofB cell function, and secretion of a number of factors aiding andabetting this response.

The involvement of the MHC Class II proteins in autoimmune disease hasbeen shown in animal models. Administration of antibodies to either MHCClass II proteins themselves or antibodies to agents that induceexpression of the MHC Class II genes interferes with development of theautoimmune condition in these model systems. The role of helper T cellshas also been demonstrated in these models by counteracting theautoimmune system using anti-CD4 monoclonal antibodies; CD4 is thecharacteristic helper T cell receptor (Shizuru, J. A. et al., Science(1988) 240:659-662).

Recent experiments have shown that, under certain circumstances, anergyor nonresponsiveness can be induced in autoreactive lymphocytes (See,Schwartz, Cell (1989) 1073-1081, which is incorporated herein byreference). In vitro experiments suggest that antigen presentation byMHC Class II molecules in the absence of an unknown co-stimulatorysignal induces a state of proliferative non-responsiveness in syngeneicT cells (Quill et al., J. Immuno. (1987) 138:3704-3712, which isincorporated herein by reference). These reports, however, provide noclear evidence that induction of anergy in vivo is possible or thatautoimmune disease can be effectively treated in this manner.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions that canbe used to identify and inhibit those aspects of the immune system whichare responsible for undesirable autoimmunity. The invention compositionsand methods are designed to target helper T cells which recognize aparticular antigen in association with a glycoprotein encoded by theMHC. The invention complexes effectively substitute for theantigen-presenting cell and cause non-responsiveness in autoreactiveT-lymphocytes and other cells of the immune system.

The invention provides forms of an autoantigen which interact with theimmune system, in a manner analogous to those initiated by theautoantigen itself to cause the autoimmune reaction. Compositions of thepresent invention are purified two component complexes of (1) aneffective portion of the MHC-encoded antigen-presenting glycoprotein;and (2) an effective portion of the antigen. These two components may bebound covalently or by noncovalent association. Evidence from both invitro and in vivo experiments establishes that such complexes induceclonal anergy in syngeneic T cells.

In other aspects, the invention is directed to pharmaceuticalcompositions wherein the complexes of the invention are activeingredients. The compositions can be used to down-regulate parts of theimmune system reactive with a particular self-antigen associated with anautoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a typical complex of the invention.

FIG. 2 shows the 3-dimensional structure of the human HLA-A2 antigen(Class I).

FIG. 3 shows a diagrammatic representation of the active portion of amodified Class II MHC-encoded glycoprotein.

FIG. 4 shows preferred second generation MHC protein designs.

FIG. 5 is a diagram of a planar membrane bilayer including the MHCglycoprotein, mimicking the surface of the antigen presenting cell.

FIG. 6 shows the amino acid sequence and encoding MRNA for the alphasubunit of acetylcholine receptor protein.

FIG. 7 shows the amino acid sequence of myelin basic protein.

FIG. 8 shows the nucleotide sequence encoding the I-A^(b) -alpha chain.

FIG. 9 shows the nucleotide sequence encoding the I-A^(b) -beta chain.

FIG. 10 presents a list of the DQ/DR haplotypes in humans and theirassociations with autoimmune diseases.

FIG. 11 shows a protocol suitable for the utilization of the complexesof the invention for the diagnosis and/or treatment of an autoimmunedisease.

FIG. 12 shows a scheme for the preparation of I-A^(k) containing NP-40soluble membrane extracts.

FIG. 13A is a scheme for the affinity purification of 10-2.16 monoclonalantibody and its coupling to CNBr activated Sepharose 4B.

FIG. 13B is a copy of a gel showing the purity of 10-2.16 monoclonalantibody purified by the scheme in FIG. 13A.

FIG. 14 shows a scheme for the purification of I-A^(k).

FIG. 15 is a polyacrylamide gel showing the purity of I-A^(k) purifiedby the scheme in FIG. 14.

FIG. 16 is a bar graph showing the results of eight studies on theinhibition of proliferation by a complex containing I-A^(k) and MBP(1-13).

FIG. 17 is a graph showing the development of EAE in mice resulting fromimmunization with MBP (1-13).

FIG. 18 is a graph showing the adoptive transfer of EAE by T cell clone4R3.4, obtained from B10A(4R) strain of mice following immunization withMBP(1-11).

FIGS. 19A-C show treatment of passively induced EAE in mice using PBSalone (19a), I-A^(s) /MBP1-14 (a non-encephalitogenic peptide) (19b),and I-A^(s) /MBp91-103 (19c).

FIG. 20 shows that complexes of the invention exist as aggregatesbecause they pass through the column with the void volume and thus havea molecular weight greater than 600,000.

FIG. 21 shows that complexes of the invention (MHC II:AChRα 100-116)were effective in treating MG in rats.

FIGS. 22A and 22B show that cell death follows the induction of anergyin T cells.

FIGS. 23A and 23B show that the molar concentration of complex of theinvention required to induce anergy is much less than that of peptidealone.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides complexes which can be used to modulate Tcell function. For instance, the complexes can be used to inhibit adeleterious T cel-mediated immune response, such as allergic responses,allograft rejection, and autoimmune diseases. In addition, the complexesof the invention can also be used as vaccines and thus, promote T cellresponses.

The invention complexes contain at least two components: a peptide whichrepresents an autoantigen or other antigenic sequence with the relevanteffect on the immune system and an effective portion of the MHC-encodedglycoprotein involved in antigen presentation. An effective portion ofan MHC glycoprotein is one which comprises the antigen binding sites andsequences necessary for recognition of the MHC-peptide complex by theappropriate T cell receptor. The MHC component can be either a Class Ior a Class II molecule. The association between the peptide antigen andthe antigen binding sites of the MHC protein can be by covalent or bynoncovalent bonding.

In other embodiments the complexes may also contain an effectorcomponent which is generally a toxin or a label. The effector portionmay be conjugated to either the MHC-encoded glycoprotein or to theautoantigenic peptide. Complexes containing an effector component aredisclosed and claimed in copending application U.S. Ser. No. 07/367,751filed Jun. 21, 1989, supra.

Each of the components of the system is described separately below;followed by description of the methods by which these complexes can beprepared, evaluated and employed.

The MHC-Derived Component

The glycoproteins encoded by the MHC have been extensively studied inboth the human and murine systems. In general, they have been classifiedas Class I glycoproteins, found on the surfaces of all cells andprimarily recognized by cytotoxic T cells; and Class II which are foundon the surfaces of several cells, including accessory cells such asmacrophages, and are involved in presentation of antigens to helper Tcells. Some of the histocompatibility proteins have been isolated andcharacterized. For a general review of MHC glycoprotein structure andfunction, see Fundamental Immunology, 2d Ed., W. E. Paul, ed., RavensPress N.Y. 1989, which is incorporated herein by reference. The term"isolated MHC component" as used herein refers to an MHC glycoprotein oran effective portion of an MHC glycoprotein (.i.e., one comprising anantigen binding site or sites and sequences necessary for recognition bythe appropriate T cell receptor) which is in other than its nativestate, for example, not associated with the cell membrane of a cell thatnormally expresses MHC. As described in detail below, the MHC componentmay be recombinantly produced, solubilized from the appropriate cellsource or associated with a liposome.

Methods for purifying the murine I-A (Class II) histocompatibilityproteins have been disclosed by Turkewitz, A. P., et al., MolecularImmunology (1983) 20:1139-1147, which is incorporated herein byreference. These methods, which are also suitable for Class I molecules,involve preparation of a soluble membrane extract from cells containingthe desired MHC molecule using nonionic detergents, such as NP-40, Tween80 and the like. The MHC molecules are then purified by affinitychromatography, using a column containing antibodies raised against thedesired MHC molecule. Use of 0.02% Tween-80 in the elution buffer ishelpful to eliminate aggregation of the purified molecules.

The isolated antigens encoded by the I-A and I-E subregions have beenshown to consist of two noncovalently bonded peptide chains: an alphachain of 32-38 kd and a beta chain of 26-29 kd. A third, invariant, 31kd peptide is noncovalently associated with these two peptides, but itis not polymorphic and does not appear to be a component of the antigenson the cell surface (Sekaly, R. P., J. Exp. Med. (1986) 164:1490-1504,which is incorporated herein by reference). The alpha and beta chains ofseven allelic variants of the I-A region have been cloned and sequenced(Estees, "T cell Clones", 3-19).

The human Class I proteins have also been studied. The MHC of humans(HLA) on chromosome 6 has three loci, HLA-, HLA-B, and HLA-C, the firsttwo of which have a large number of alleles encoding alloantigens. Theseare found to consist of a 44 kd subunit and a 12 kd beta₂ -microglobulinsubunit which is common to all antigenic specificities. Isolation ofthese detergent-soluble HLA antigens was described by Springer, T. A.,et al., Proc. Natl. Acad. Sci. USA (1976) 73:2481-2485; Clementson, K.J., et al., in "Membrane Proteins" Azzi, A., ed; Bjorkman, P., Ph.D.Thesis Harvard (1984) all of which are incorporated herein by reference.

Further work has resulted in a detailed picture of the 3-D structure ofHLA-A2, a Class I human antigen. (Bjorkman, P. J., et al., Nature (1987)329:506-512, 512-518 which is incorporated herein by reference). In thispicture, the β₂ -microglobulin protein and alpha₃ segment of the heavychain are associated; the alpha₁ and alpha₂ regions of the heavy chainappear to form antigen-binding sites to which the peptide is bound(Science (1987) 238:613-614, which is incorporated herein by reference)Bjorkman, P. J. et al. Nature (supra). Soluble HLA-A2 can be purifiedafter papain digestion of plasma membranes from the homozygous humanlymphoblastoid cell line J-Y as described by Turner, M. J. et al., J.Biol. Chem. (1977) 252:7555-7567, all of which are incorporated hereinby reference. Papain cleaves the 44 kd chain close to the transmembraneregion yielding a molecule comprised of alpha₁, alpha₂, alpha₃, and β₂microglobulin. A representation of the deduced three dimensionalstructure of the Class I HLA-A2 antigen is shown in FIG. 2.

While the three dimensional structure of Class II MHC antigens is notknown in such detail, it is thought that Class II glycoproteins have adomain structure, including an antigen binding site, similar to that ofClass I. It is formed from the N-terminal domain portions of two classII chains which extend from the membrane bilayer. The N-terminal portionof one chain has two domains of homology with the alpha₁ and alpha₂regions of the MHC Class I antigen sequence. Cloning of the Class IIgenes (as described by Estees supra) permits manipulation of the ClassII MHC binding domains for example, as described below.

The MHC glycoprotein portions of the complexes of the invention, then,can be obtained by isolation from lymphocytes and screened for theability to bind the desired peptide antigen. The lymphocytes are fromthe species of individual which will be treated with the complexes. Forexample, they may be isolated from human B cells from an individualsuffering from the targeted autoimmune disease, which have beenimmortalized by transformation with a replication deficient Epstein-Barrvirus, utilizing techniques known in the art.

MHC glycoproteins have been isolated from a multiplicity of cells usinga variety of techniques including solubilization by treatment withpapain, by treatment with 3M KCl, and by treatment with detergent. In apreferred method detergent extraction of Class II protein fromlymphocytes followed by affinity purification is used. Detergent canthen be removed by dialysis or selective binding beads, e.g., Bio Beads.

Alternatively, the amino acid sequence of each of a number of Class IIproteins are known, and the genes have been cloned, therefore, theproteins can be made using recombinant methods. In a first generationsynthetic MHC protein, the heavy (alpha) and light (beta) chains aresynthesized using a carboxy terminal truncation which effects thedeletion of the hydrophobic domain, and the carboxy termini can bearbitrarily chosen to facilitate the conjugation of toxins or label. Forexample, in the MHC protein shown in FIG. 3, lysine residues areintroduced. In addition, cysteine residues near the carboxy termini areincluded to provide a means to form disulfide linkage of the chains; thesynthetic gene can also include restriction sites to aid in insertioninto expression vectors and in manipulating the gene sequence to encodeanalogs. The alpha and beta chains are then inserted into expressionvectors, expressed separately in an appropriate host, such as E. coli,yeast, or other suitable cells, and the recombinant proteins obtainedare recombined in the presence of the peptide antigen.

As the availability of the gene permits ready manipulation of thesequence, a second generation of preferred construction includes hybridClass I and Class II features, as illustrated in FIG. 4, wherein thealpha₁ and beta₁ domains of Class II MHC are linked through a flexibleportion that permits intramolecular dimerization between these domainsresulting in an edge-to-edge beta sheet contact. The beta₁ segment isthen fused to the alpha₂ domain of Class I with beta₂ microglobulincoexpressed to stabilize the complex. The transmembrane andintracellular domains of the Class I gene can also be included but theremay be no point in doing so unless liposomes are used to transport thecomplex. A simpler version includes only the alpha₁ and beta₁ domainswith a C-terminal lysine for toxin conjugation (FIG. 4).

Construction of expression vectors and recombinant production from theappropriate DNA sequences are performed by methods known in the art perse. Expression can be in procaryotic or eucaryotic systems. Procaryotesmost frequently are represented by various strains of E. coli. However,other microbial strains may also be used, such as bacilli, for exampleBacillus subtilis, various species of Pseudomonas, or other bacterialstrains. In such procaryotic systems, plasmid vectors which containreplication sites and control sequences derived from a speciescompatible with the host are used. For example, E. coli is typicallytransformed using derivatives of pBR322, a plasmid derived from an E.coli species by Bolivar et al., Gene (1977) 2:95. Commonly usedprocaryotic control sequences, which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, including such commonly usedpromoters as the beta-lactamase (penicillinase) and lactose (lac)promoter systems (Change et al., Nature (1977) 198:1056) and thetryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8:4057 ) and the lambda-derived P_(L) promoter and N-generibosome binding site (Shimatake et al., Nature (1981) 292:128). Anyavailable promoter system compatible with procaryotes can be used. Allreferences cited herein whether supra or infra, are hereby incorporatedherein by reference.

The expression systems useful in the eucaryotic hosts comprise promotersderived from appropriate eucaryotic genes. A class of promoters usefulin yeast, for example, include promoters for synthesis of glycolyticenzymes, including those for 3-phosphoglycerate kinase (Hitzeman, etal., J. Biol. Chem. (1980) 255:2073). Other promoters include those fromthe enolase gene (Holland, M. J., et al. J. Biol. Chem. (1981) 256:1385)or the Leu2 gene obtained from YEp13 (Broach, J., et al., Gene (1978)8:121).

Suitable mammalian promoters include the early and late promoters fromSV40 (Fiers, et al., Nature (1978) 273:113) or other viral promoterssuch as those derived from polyoma, adenovirus II, bovine papillomavirus or avian sarcoma viruses. Suitable viral and mammalian enhancersare cited above.

The expression system is constructed from the foregoing control elementsoperably linked to the MHC sequences using standard methods, employingstandard ligation and restriction techniques which are well understoodin the art. Isolated plasmids, DNA sequences, or synthesizedoligonucleotides are cleaved, tailored, and relegated in the formdesired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer or these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 ug ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ulof buffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about 1 hr to 2 hr at about 37° C. areworkable, although variations can be tolerated. After each incubation,protein is removed by extraction with phenol/chloroform, and may befollowed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol followed by running overa Sephadex G-50 spin column. If desired, size separation of the cleavedfragments may be performed by polyacrylamide gel or agarose gelelectrophoresis using standard techniques. A general description of sizeseparation is found in Methods in Enzymology (1980) 65:499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° to 25° C. in 50 Mm Tris Ph 7.6, 50 Mm NaCl, 6mM MgCl₂, 6 Mm DTT and 5-10 uM dNTPs. The Klenow fragment fills in a 5'sticky ends but chews back protruding 3' single strands, even throughthe four dNTPS, are present. If desired, selective repair can beperformed by supplying only one of the, or selected, dNTPs within thelimitations dictated by the nature of the sticky ends. After treatmentwith Klenow, the mixture is extracted with phenol/chloroform and ethanolprecipitated followed by running over a Sephadex G-50 spin column.

Synthetic oligonucleotides are prepared using commercially availableautomated oligonucleotide synthesizers. Kinasing of single strands priorto annealing or for labeling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 0.1 nmole substratein the presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol,1-2 mMATP, 1.7 pmoles ³² P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1mM EDTA.

Ligations are performed in 15-30 ul volumes under the following standardconditions and temperatures: 20 mM Tris-HCl pH 7.5, 10 mMMgCl₂, 10 mMDTT, 33 ug/ml BSA, 10 mM-50 mM NaCl, and either 40 uM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for "sticky end" ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 ug/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 uM total endsconcentration.

In vector construction employing "vector fragments," the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) in orderto remove the 5' phosphate and prevent religation of the vector. BAPdigestions are conducted at pH 8 in approximately 150 mM Tris, in thepresence of Na⁺ and Mg⁺² using about 1 unit of BAP per ug of vector at60° C. for about 1 hr. In order to recover the nucleic acid fragments,the preparation is extracted with phenol/chloroform and ethanolprecipitated and desalted by application to a Sephadex G-50 spin column.Alternatively, religation can be prevented in vectors which have beendouble digested by additional restriction enzyme digestion of theunwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis can beused. This is conducted using a primer synthetic oligonucleotidecomplementary to a single stranded phage DNA to be mutagenized exceptfor limited mismatching, representing the desired mutation. Briefly, thesynthetic oligonucleotide is used as a primer to direct synthesis of astand complementary to the phage, and the resulting double-stranded DNAis transformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells which harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The resulting plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

In the proteins of the invention, however, a synthetic gene isconveniently employed. The gene design can include restriction siteswhich permit easy manipulation of the gene to replace coding sequenceportions with these encoding analogs.

Correct ligations for plasmid construction can be confirmed by firsttransforming E. coli strain MM294 obtained from E. coli Genetic StockCenter, CGSC #6135, or other suitable host with the ligation mixture.Successful transformants are selected by ampicillin, tetracycline orother antibiotic resistance or using other markers depending on the modeof plasmid construction, as is understood in the art. Plasmid from thetransformants are then prepared according to the method of Clewell, D.B., et al., Proc. Natl. Acad. Sci. USA (1969) 62:1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., J. Bacteriol.(1972) 110:667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al., Proc. Natl. Acad.Sci. USA (1977) 74:5463 as further described by Messing, et al., NucleicAcids Res. (1981) 9:309, or by the method of Maxam, et al., Methods inEnzymology (1980) 65:499.

The constructed vector is then transformed into a suitable host forproduction of the protein. Depending on the host cell used,transformation is done using standard techniques appropriate to suchcells. The calcium treatment employing calcium chloride, as described byCohen, S. N., Proc. Natl. Acad. Sci. USA (1972) 69:2110, or the RbClmethod described in Maniatis, et al., Molecular Cloning: A LaboratoryManual (1982) Cold Spring Harbor Press, p. 254 is used for procaryotesor other cells which contain substantial cell wall barriers. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology (1978) 52:546 orelectroporation is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P., et al., J. Bacter. (1977)130:946 and Hsiao, C. L., et al., Proc. Natl. Acad. Sci. USA (1979)76:3829.

The transformed cells are then cultured under conditions favoringexpression of the MHC sequence and the recombinantly produced proteinrecovered from the culture.

Antigenic Peptides

The autoantigenic proteins or tissues for a number of autoimmunediseases are known. For example, in experimentally induced autoimmunediseases, antigens involved in pathogenesis have been characterized: inarthritis in rat and mouse, native type-II collagen is identified incollagen-induced arthritis, and mycobacterial heat shock protein inadjuvant arthritis (Stuart et al. (1984), Ann. Rev. Immunol. 2:199-218;van Eden et al. (1988), Nature 331:171-173.); thyroglobulin has beenidentified in experimental allergic thyroiditis (EAT) in mouse (Maron etal. (1988), J. Exp. Med. 152:1115-1120); acetyl choline receptor (AChR)in experimental allergic myasthenia gravis (EAMG) (Lindstrom et al.(1988), Adv. Immunol. 42:233-284); and myelin basic protein (MBP) andproteolipid protein (PLP) in experimental allergic encephalomyelitis(EAE) in mouse and rat (See Acha-Orbea et al., supra). In addition, forexample, target antigens have been identified in humans: type-IIcollagen in human rheumatoid arthritis (Holoshitz et al. (1986), Lancetii:305-309); and acetyl choline receptor in myasthenia gravis (Lindstromet al. (1988), supra) all of the above are incorporated herein byreference.

It is believed that the presentation of antigen by the MHC glycoproteinon the surface of antigen-presenting cells (APCs) occurs subsequent tothe hydrolysis of antigenic proteins into smaller peptide units. Thelocation of these smaller segments within the antigenic protein can bedetermined empirically. These segments are thought to be 8-15 residuesin length, and contain both the agretope (recognized by the MHCmolecule) and the epitope (recognized by T cell receptor on the T-helpercell). The epitope itself is a contiguous or non-contiguous sequence of5-6 amino acids which recognizes the antigen-specific receptor ofT-helper cells. The agretope is a continuous or non-contiguous sequencewhich is responsible for the association of the peptide with the MHCglycoproteins.

The empirical process of determining the relevant 8-15 amino acidsubunits is illustrated using the alpha subunit of the acetylcholinereceptor of skeletal muscle. In myasthenia gravis (MG) an autoimmuneresponse is directed to a region of this subunit. A loss of the acetylcholine receptors on the postsynaptic membrane of the neuromuscularjunction causes the MG symptoms.

In MG, autoantibodies against the alpha subunit of the acetylcholinereceptor (AChR) are associated with the autoimmune response directed atthe AChR. Eighty five percent of MG patients have autoantibodiesreactive with the alpha subunit. Of these, 60% have antibodies that bindto a peptide segment of the alpha subunit called the main immunogenicregion (MIR) which is located between residues 60 and 80 (Tzartos andLindstrom, Proc. Natl. Acad. Sci. USA (1980) 77:755). The peptidesegments recognized by autoreactive human T cells also are located onthe alpha subunit (Hohfield, et al., Proc. Natl. Acad. Sci. USA (1987).The epitopes recognized by these T cells lie between residues 1-30,125-147, 169-181, 257-271 and 351-368. In addition, in humans the AChRpeptides 195-212 and 257-269 have been partially characterized asepitopes in myasthenia gravis patients of the HLA-DR5 and HLA-DR3, DQw2MHC haplotypes, respectively (See Acha-Orbea (1989), supra).

The peptides carrying agretopes permitting presentation of the epitopesassociated with alpha subunit of this receptor are readily determined.For example, determination of the appropriate peptides in a mouse modelis carried out as follows. Strains of mice which, when immunized withTorpedo californicus AChR develop a disease with many of the features ofhuman myasthenia gravis, are used as model. MHC Class II glycoproteinsare isolated from spleen cells of mice of this strain using lectin andmonoclonal antibody affinity supports. The purified MHC Class IIproteins are incorporated into phospholipid vesicles by detergentdialysis. The resultant vesicles are then allowed to fuse to clean glasscover slips to produce on each a planar lipid bilayer containing MHCmolecules as shown in FIG. 5 (Brian and McConnell, Proc. Natl. Acad.Sci. USA (1984) 81:6159, which is incorporated herein by reference).

One cover slip containing MHC Class II molecules embedded in theadherent planar lipid membrane is placed in each well of several 24-wellculture plates. Each one of the approximately 40 overlapping 20-residuesynthetic peptides corresponding to the alpha subunit sequence andcontaining one or more radiolabeled amino acid residues (prepared asdescribed below) is placed in a well with cover slip and PBS and allowedto incubate several days. The extent of binding of peptide in the MHCClass II glycoprotein antigen binding site is measured by the amount ofradio-activity incorporated into the MHC Class II-planar lipid membraneon the cover slip versus planar lipid membrane alone. Specificincorporation of radioactivity indicates that the bound peptide containsan agretope (MHC Class II peptide binding site) of one of the severalspecies of MHC Class II molecules present in the planar lipid membrane.In this way, the set of agretopes for the alpha subunit of AChR isdefined for the mouse strain that displays the symptoms of MG uponimmunization with AChR or purified alpha subunit.

Next, each of the alpha subunit synthetic peptide segments that containan agretope is again incorporated into the antigen binding site ofisolated MHC Class II proteins embedded in planar lipid membranes oncover slips. One cover slip is added to each well of a 24-well cultureplate, and spleen cells from mice immunized against AChR (and from whichstrain the adherent MHC Class II proteins were isolated) are added toeach well. T cell hybridoma proliferation, as measured by tritiatedthymidine uptake into DNA, indicates that the MHC Class II protein-boundpeptide contains both an agretope and an epitope for binding to the Tcell. Activation of T cell clones is determined by measuring IL-3production (see, Quill et al., supra).

The Dupont apparatus and technique for rapid multiple peptide synthesis(RAMPS) is used to synthesize the members of a set of overlapping (10residue overlap), 20-residue peptides from the alpha subunit of Torpedocalifornicus AChR. The sequence of this peptide is known and is shown inFIG. 6. One or more radioactive amino acids is incorporated into eachsynthetic peptide. The pentafluorphenyl active esters of sidechain-protected, FMOC amino acids are used to synthesize the peptides,applying standard stepwise solid phase peptide synthetic methods,followed by standard side chain deprotection and simultaneous release ofthe peptide amide from the solid support.

Alternatively the overlapping sequences which include the putativesegments of 8-15 amino acids of the antigenic protein, such asacetylcholine receptor protein, can be synthesized on the method ofGeysen, H. M., et al. J. Immun. Meth. (1987) 102:274, which isincorporated herein by reference. The synthesized radio labeled peptidesare tested by incubating them individually (on the plates) with purifiedMHC proteins which have been formulated into lipid membrane bilayers asabove.

In multiple sclerosis (MS), which results in the destruction of themyelin sheath in the central nervous system, myelin basic protein (MBP),the major protein component of myelin is the principal autoantigen.Pertinent segments of the MBP protein are also determined empirically,using a strain of mice which develops experimental allergic encephalitis(EAG) when immunized with bovine myelin basic protein, the sequence ofMBP is shown in FIG. 7.

Systemic lupus erythematosus (SLE) has a complex systemology, butresults from an autoimmune response to red blood cells. Peptides whichare the antigenic effectors of this disease are found in the proteins onthe surface of red blood cells.

Rheumatoid arthritis (RA) is a chronic inflammatory disease resultingfrom an immune response to proteins found in the synovial fluid.

Insulin-dependent diabetes mellitus (IDDM) results from autoimmuneattack on the beta cells within the Islets of Langerhans which areresponsible for secretion of insulin. Circulating antibodies to Isletscells surface antigens and to insulin are known to precede IDDM.Critical peptides in eliciting the immune response in IDDM are believedto be portions of the insulin sequence and the beta cell membranesurface proteins.

The relevant antigenic peptide subunits, as they are relatively short,can readily by synthesized using standard automated methods for peptidesynthesis. In the alternative, they can be made recombinantly usingisolated or synthetic DNA sequences; though this is not the mostefficient approach for peptides of this length.

Thus, in summary, a set of labeled test peptides is prepared, and thosewhich bind to MHC in planar lipid membranes containing MHC proteins areshown to contain the agretope.

The identified peptides are then prepared by conventional solid phasesynthesis and the subset which contain epitopes for the disease-inducinghelper T cell clones is determined by incubation of the candidatepeptides with murine antigen-presenting cells (APC) (or with isolatedMHC complex) and spleen or lymph node T cells from mice immunized withthe full length protein. Successful candidates will stimulate T cellproliferation in this system. This second, smaller, subset representsthe suitable peptide component.

Formation of the Complex

The elements of the complex can be associated by standard means known inthe art. The antigenic peptides can be associated noncovalently with thepocket portion of the MHC protein by, for example, mixing the twocomponents. They can also be covalently bound using standard proceduresby, for example, photo affinity labelling, (see e.g., Hall et al.,Biochemistry 24:5702-5711 (1985), which is incorporated herein byreference).

For example, the AChR peptide 195-215, which has been characterized asan epitope in MG in humans and in mice, may be connected to theN-terminal antigen binding site of a polypeptide derived from an MHCantigen associated with MG. The amino acid sequence of the AChR peptidein one letter amino acid code is:

    DTPYLDITYHFIMQRIPLYFV

An oligonucleotide which encodes the peptide is synthesized using theknown codons for the amino acid, preferably those codons which havepreferred utilization in the organism which is to be used for expressionare utilized in designing the oligonucleotide. Preferred codonutilizations for a variety of organisms and types of cells are known inthe art. If, for example, expression is to be in E. coli, a suitableoligonucleotide sequence encoding AChR 195-215 could be:

    5' GAC ACC CCG TAC CTG GAC ATC ACC TAC CAC TTC ATC ATG CAG CGT ATC CCG CTG TAC TTC CTG 3'.

This sequence may then be incorporated into a sequence encoding thepeptides derived from the MHC antigen, utilizing techniques known in theart. The incorporation site will be such that, when the molecule isexpressed and folded, the AChR peptide antigen will be available as anepitope for the target T cells.

In one protocol, the AChR 195-215 peptide is attached to the N-terminalend of the appropriate MHC molecule. If the recombinant complex is to beused in mice, for example, the AChR peptide may be incorporated into asequence encoding either the I-A^(b) -alpha or I-A^(b) -beta chain. Thesequences encoding these chains are known, and are shown in FIG. 8(alpha chain), and FIG. 9 (beta chain); also shown in the figures arerestriction enzyme sites and significant domains of the chains. If theAChR peptide is to be incorporated into the beta chain, for example, theoligonucleotide may be inserted as a replacement for the leader peptide.Methods of replacing sequences within polynucleotides are known in theart, examples of which are described in the section on the constructionof plasmids.

A similar protocol may be used for incorporation of the AChR peptideinto a sequence encoding a peptide derived from the appropriate humanHLA antigen. For example, in humans, the haplotype DR2W2 is associatedwith MG. Hence, the AChR peptide may be incorporated into, for example,a sequence encoding a beta-chain of a DR2 allele. The structural basisin the DR subregion for the major serological specificities DR1-9 areknown, as are the sequences encoding the HLA-DR-beta chains from anumber of DR haplotypes. See, for e.g., Bell et al. (1987), Proc. Natl.Acad. Sci. USA 84:6234-6238 which are incorporated herein by reference.

As demonstrated above, the autoimmune antigen peptide and the MHCcomponent may be linked via peptide linkages. However, other modes oflinkage are obvious to those of skill in the art, and could include, forexample, attachment via carbohydrate groups on the glycoproteins,including, e.g., the carbohydrate moieties of the alpha-and/orbeta-chains.

Assessment of the Complex

The complexes of the invention can be assayed using an in vitro systemor using an in vivo model. In the in vitro system, the complex isincubated with peripheral blood T cells from subjects immunized with, orshowing immunity to, the protein or antigen responsible for thecondition associated with the peptide of the complex. The successfulcomplexes will induce anergy in syngeneic T cells and preventproliferation of the T cells even upon stimulation with additionalantigen.

In the in vivo system, T cells that proliferate in response to theisolated epitope or to the full length antigen in the presence of APCare cloned. The clones are injected into histocompatible animals whichhave not been immunized in order to induce the autoimmune disease.Symptoms related to the relevant complex should ameliorate or eliminatethe symptoms of the disease.

Either of the types of complexes, i.e., with or without the effectorcomponent, may be used. In one mode the treatment is two-fold. Theindividual is treated with the complex of MHC-encoded antigen-presentingglycoprotein containing an effective portion of the antigen todown-regulate the immune system. Further down-regulation is achieved bytreatment with the three component complex with includes the MHC-encodedantigen-presenting glycoprotein, an effective portion of antigen whichis specific for the autoimmune disease being treated, and an effectorcomponent. In addition, panels of complexes may be used for treatment.For example, if it is suspected that more than one peptide of an antigenis involved in the autoimmune response, and/or if it is suspected thatmore than one antigen is involved, the individual may be treated withseveral complexes selected from a panel containing the effective portionof the appropriate MHC-encoded antigen-presenting polypeptides, andeffective portions of antigens; these may be with or without effectorcomponents.

Administration of a labeled complex permits identification of thoseportions of the immune system involved in the disease, in diagnosticapplications.

Selection of the MHC Complexes for Therapy and/or Diagnosis

In order to select the MHC complexes of the invention which are to beused in the diagnosis or treatment of an individual for an autoimmunedisease, the type of MHC antigens which are involved in the presentationof the autoantigen are identified.

Specific autoimmune dysfunctions are correlated with specific MHC types.A list of the DQ/DR haplotypes in humans and their associations withautoimmune diseases are shown in FIG. 10. Methods for identifying whichalleles, and subsequently which MHC encoded polypeptides, are associatedwith an autoimmune disease are known in the art. A method described inEP 286447 is suitable. In this method several steps are followed. First,the association between an MHC antigen and the autoimmune disease isdetermined based upon genetic studies. The methods for carrying outthese studies are known to those skilled in the art, and information onall known HLA disease associations in humans is maintained in the HLAand Disease Registry in Copenhagen. The locus encoding the polypeptideassociated with the disease is the one that would bear the strongestassociation with the disease (See FIG. 10).

Second, specific alleles encoding the disease associated with MHCantigen/polypeptide are identified. In the identification of thealleles, it is assumed that the susceptibility allele is dominant.Identification of the allele is accomplished by determining the strongpositive association of a specific subtype with the disease. This may beaccomplished in a number of ways, all of which are known to thoseskilled in the art. E.g., subtyping may be accomplished by mixedlymphocyte response (MLR) typing and by primed lymphocyte testing (PLT).Both methods are described in Weir and Blackwell, eds., Handbook ofExperimental Immunology, which is incorporated herein by reference. Itmay also be accomplished by analyzing DNA restriction fragment lengthpolymorphism (RFLP) using DNA probes that are specific for the MHC locusbeing examined. E.g., Nepom (1986), Annals N.Y. Acad. Sci. 475, 1.Methods for preparing probes for the MHC loci are known to those skilledin the art. See, e.g., Gregersen et al. (1986), Proc. Natl. Acad. Sci.USA 79:5966; Weissman et al. in Medicine in Transition: the Centennialof the University of Illinois College of Medicine (E. P. Cohen, ed.1981) all of which are incorporated herein by reference.

The most complete identification of subtypes conferring diseasesusceptibility is accomplished by sequencing of genomic DNA of thelocus, or cDNA to mRNA encoded within the locus. The DNA which issequenced includes the section encoding the hypervariable regions of theMHC encoded polypeptide. Techniques for identifying specifically desiredDNA with a probe, for amplification of the desired region are known inthe art, and include, for example, the polymerase chain reaction (PCR)technique.

Once the allele which confers susceptibility to the specific autoimmunedisease is identified, the polypeptide encoded within the allele is alsoidentifiable, i.e., the polypeptide sequence may be deduced from thesequence of DNA within the allele encoding it. The MHC antigen complexesof the invention used for diagnosis and/or therapy are derived from theeffective portion of the MHC antigen associated with the autoimmunedisease state and from an autoimmune antigen associated with the samedisease state.

As an example, over 90% of rheumatoid arthritis patients have ahaplotype of DR4(Dw4), DR4(Dw14) or DR1 (See FIG. 10). It is also knownthat a target antigen in human rheumatoid arthritis is type-II collagen.Hence, the complexes of the invention used for treatment or diagnosis ofan individual with rheumatoid arthritis would include those containing apolypeptide derived from the DR4(Dw4), DR1 and/or DR4(Dw14) which iscapable of antigen presentation for disease induction, or incapable ofantigen presentation for disease suppression, complexed with aneffective portion of type-II collagen.

A protocol which may be suitable for the utilization of the complexes ofthe invention for the diagnosis and/or treatment of an autoimmunedisease is depicted in FIG. 11. Briefly, an individual having (orsusceptible to) an autoimmune disease is identified, and the autoimmunedysfunction is identified. Identification may be by symptomology and/oran examination of family histories. The individual's MHC type isdetermined by one or more of several methods known in the art,including, for example, cell typing by MLR, by serologic assay, and byDNA analysis (including RFLP and PCR techniques). The individuals Tcells are examined in vitro, to determine the autopeptide(s) recognizedby autoreactive T cells; this is accomplished utilizing labeledcomplexes of the invention, described supra, which are of the formula X¹MHC² peptide, wherein X is a label moiety. After it is determined whichcomplexes target the T cells, the individual is treated with complexesof the invention which are able to suppress the specific autoreactive Tcell replication and/or those which kill the autoreactive T cells; theseare complexes of the type MHC² peptide, and, X¹ MHC² peptide (wherein Xis a moiety capable of killing the T cell), respectively. Therapy (asdetermined by the autoreactive T cells remaining) is monitored with Tcell binding studies using the labeled complexes of the invention,described supra.

As used herein, the term "individual" encompasses all mammals and allvertebrates which possess basically equivalent MHC systems.

Model Systems for In vivo Testing

The following are model systems for autoimmune diseases which can beused to evaluate the effects of the complexes of the invention on theseconditions.

Systemic Lupus Erythematosus (SLE)

F₁ hybrids of autoimmune New Zealand black (NZB) mice and thephenotypically normal New Zealand White (NZW) mouse strain developsevere systemic autoimmune disease, more fulminant than that found inthe parental NZB strain. These mice manifest several immuneabnormalities, including antibodies to nuclear antigens and subsequentdevelopment of a fatal, immune complex-mediated glomerulonephritis withfemale predominance, remarkably similar to SLE in humans. Knight, etal., J. Exp. Med. (1978) 147:1653, which is incorporated hereby byreference.

In both the human and murine forms of the disease, a strong associationwith MHC gene products has been reported. HLA-DR2 and HLA-DR3individuals are at a higher risk than the general population to developSLE (Reinertsen, et al., N. Engl. J. Med (1970) 299:515), while in NZB/WF₁ mice (H-2^(d/u)), a gene linked to the h-2^(u) haplotype derived fromthe NZW parent contributes to the development of the lupus-likenephritis.

The effect of the invention complex can be measured by survival ratesand by the progress of development of the symptoms, such as proteinuriaand appearance of anti-DNA antibodies.

Proteinuria is measured calorimetrically by the use of Uristix (MilesLaboratories, Inc., Elkhart, Ind.), giving an approximation ofproteinuria as follows: trace, 10 mg/dl; 1+, 30 mg/dl; 100 mg/dl; 3+,300 mg/dl; and 4+, 1000 mg/dl. The development of high grade proteinuriais significantly delayed by treatment of the mice with complex.

The presence of anti-DNA specific antibodies in NZB/W F₁ mice isdetermined by using a modification of a linked immunosorbent assay(ELISA) described by Zouali and Stollar, J. Immunol. Methods (1986)90:105 which is incorporated herein by reference.

Myasthenia Gravis (MG)

Myasthenia gravis is one of several human autoimmune diseases linked toHLA-D. Safenberg, et al., Tissue Antigens (1978) 12:136; McDevitt, etal., Arth. Rheum. (1977) 20:59 which are incorporated herein byreference. In MG, antibodies to the acetyl choline receptors (AcChoR)impair neuromuscular transmission by mediating loss of AcChoR in thepostsynaptic membrane.

SJL/J female mice are a model system for human MG. In these animals,experimental autoimmune myasthenia gravis (EAMG) is induced byimmunizing the mice with soluble AcChoR protein from another species.Susceptibility to EAMG is linked in part to the MHC and has been mappedto the region within H-2. Christadoss, et al., J. Immunol. (1979)123:2540.

AcChoR protein is purified from Torpedo californica and assayedaccording to the method of Waldor, et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:2713, incorporated by reference. Emulsified AcChoR, 15 ug incomplete Freund adjuvant, is injected intradermally among six sites onthe back, the hind foot pads, and the base of the tail. Animals arereimmunized with this same regimen 4 weeks later.

Evaluation can be made by measurement of anti-AcChoR antibodies,Anti-AcChoR antibody levels are measured by a microliter ELISA assay asdescribed in Waldor, et al., supra. The standard reagent volume is 50 ulper well. Reagents are usually incubated in the wells for 2 hr at RT.Five ug of AcChoR diluted in bicarbonate buffer, pH 9.6, is added toeach well. After incubation with AcChoR, the plates are rinsed fourtimes with a wash solution consisting of phosphate-buffer salinecontaining 0.05% Tween and 0.05% NaN₃. Mouse sera are diluted in 0.01MPBS (pH 7.2), 1.5 mfr MgCl₂, 2.0 mM 2-mercaptoethanol, 0.05% Tween-80,0.05% NaN₃ (P-Tween buffer) and incubated on the plate. After the plateis washed, beta-galactosidase-conjugated sheep anti-mouse antibodydiluted in P-Tween buffer is added to each well. After a final washing,the enzyme substrate, p-nitrophenylgalctopyranoside is added to theplate, and the degree of substrate catalysis is determined from theabsorbance at 405 nm after 1 hr.

Anti-AcChoR antibodies are expected to be present in the immunized withAcChoR mice as compared to non-immunized mice. Treatment with complex isexpected to significantly reduce the titer of anti-AcChoR antibodies inthe immunized mice.

The effect of treatment with complex on clinical EAMG can also beassessed. Myasthenia symptoms include a characteristic hunched posturewith drooping of the head and neck, exaggerated arching of the back,splayed limbs, abnormal walking, and difficulty in righting. Mildsymptoms are present after a standard stress test, and should beameliorated by administration of complex after a period of time afterwhich antibody titer has fallen.

Rheumatoid Arthritis (RA)

In humans, susceptibility to rheumatoid arthritis is associated with HLAD/DR. The immune response in mice to native type II collagen has beenused to establish an experimental model for arthritis with a number ofhistological and pathological features resembling human RA.Susceptibility to collagen-induced arthritis (CIA) in mice has beenmapped to the H-2 I region, particularly the I-A subregion. Huse, etal., Fed. Proc. (1984) 43:1820.

Mice from a susceptible strain, DBA-1 are caused to have CIA bytreatment of the mice with native type II collagen, using the techniquedescribed in Wooley and Luthra, J. Immunol. (1985) 134:2366,incorporated herein by reference.

In another model, adjuvant arthritis in rats is an experimental modelfor human arthritis, and a prototype of autoimmune arthritis triggeredby bacterial antigens, Holoschitz, et al., Prospects of Immunology (CRCPress) (1986); Pearson Arthritis Rheum. (1964) 7:80. The disease theresult of a cell-mediated immune response, as evidenced by itstransmissibility by a clone of T cells which were reactive against theadjuvant (MT); the target self-antigen in the disease, based uponstudies with the same cloned cells, appears to be part(s) of aproteoglycan molecule of cartilage.

Adjuvant disease in rats is produced as described by Pearson, supra,i.e., by a single injection of Freund's adjuvant (killed tuberclebacilli or chemical fractions of it, mineral oil, and an emulsifyingagent) given into several depot sites, preferably intracutaneously orinto a paw or the base of the tail. The adjuvant is given in the absenceof other antigens.

The effect of complex treatment of manifestations of the disease aremonitored. These manifestations are histopathological, and include anacute and subacute synovitis with proliferation of synovial liningcells, predominantly a mononuclear infiltration of the articular andparticular tissues, the invasion of bone and articular cartilage byconnective tissue pannus, and periosteal new bone formation, especiallyadjacent to affected joints. In severe or chronic cases, destructivechanges occur, as do fibrous or bony ankylosis. These histopathologicalsymptoms are expected to appear in control animals at about 12 daysafter sensitization to the Freund's adjuvant.

Insulin Dependent Diabetes Mellitus (IDDM)

IDDM is observed as a consequence of the selective destruction ofinsulin-secreting cells within the Islets of Langerhans of the pancreas.Involvement of the immune system in this disease is suggested bymorphologic evidence of early infiltration of the Islets by mononuclearcells, by the detection of anti-islet cell antibodies, by the highfrequency of HLA-DR3 and -DR4 alleles in IDDM populations, and byclinical associations between IDDM and various autoimmune diseases. Ananimal model for spontaneous IDDM and thyroiditis has been developed inthe BB rat. As in humans, the rat disease is controlled in part by thegenes encoding the MHC antigens, is characterized by islet infiltration,and is associated with the presence of anti-islet antibodies. The I-Eequivalent Class II MHC antigens appear to be involved in manifestationof the autoimmune diseases in the BB rat. Biotard, et al., Proc. Natl.Acad. Sci. USA (1985) 82:6627.

In morphologic evaluation, insulitis is characterized by the presence ofmononuclear inflammatory cells within the islets. Thyroiditis ischaracterized by focal interstitial lymphocytic infiltrate within thethyroid gland, as a minimum criterion. Most severe cases show diffuseextensive lymphocytic infiltrates, disruption of acini, fibrosis, andfocal Hurthle cell change. See Biotard et al., supra.

Treatment of the BB rats with complex of the invention is expected toameliorate or prevent the manifestation of the clinical andmorphological symptoms associated with IDDM and thyroiditis.

In another spontaneous model, the NOD mouse strain (H-2K^(d) D^(b)) is amurine model for autoimmune IDDM. The disease in these animals ischaracterized by anti-islet cell antibodies, severe insulitis, andevidence for autoimmune destruction of the beta-cells. Kanazawa, et al.,Diabetologia (1984) 27:113. The disease can be passively transferredwith lymphocytes and prevented by treatment with cyclosporin-A (Ikehara,et al., Proc. Natl. Acad. Sci. USA (1985) 82:7743; Mori, et al.),Diabetologia (1986) 29:244. Untreated animals develop profound glucoseintolerance and ketosis and succumb within weeks of the onset of thedisease. Seventy to ninety percent of female and 20-30% of male animalsdevelop diabetes within the first six months of life. Breeding studieshave defined at least two genetic loci responsible for diseasesusceptibility, one of which maps to the MHC. Characterization of NODClass II antigens at both the serologic and molecular level suggest thatthe susceptibility to autoimmune disease is linked to I-A_(B).Acha-Orbea and McDevitt, Proc. Natl. Acad. Sci. USA (1907) 84:235.

Treatment of Female NOD mice with complex is expected to lengthen thetime before the onset of diabetes and/or to ameliorate or prevent thedisease.

Experimental Allergic Encephalomyelitis (EAE)

Experimental allergic encephalomyelitis (EAE) is an induced autoimmunedisease of the central nervous system which mimics in many respects thehuman disease of multiple sclerosis (MS). The disease can be induced inmany species, including mice and rats.

The disease is characterized by the acute onset of paralysis.Perivascular infiltration by mononuclear cells in the CNS is observed inboth mice and rats. Methods of inducing the disease, as well assymptomology, are reviewed in Aranson (1985) in The Autoimmune Diseases(eds. Rose and Mackay, Academic Press, Inc.) pp. 399-427, and inAcha-Orbea et al. (1989), Ann. Rev. Imm. 7:377-405.

One of the genes mediating susceptibility is localized in the MHC classII region (Moore et al. (1980), J. Immunol. 124:1815-1820). The bestanalyzed encephalitogenic protein is myelin basic protein (MBP), butother encephalitogenic antigens are found in the brain. The immunogenicepitopes have been mapped (see Acha-Orbea et al., supra.). In the PLmouse strains (H-2^(u)) two encephalitogenic peptides in MBP have beencharacterized: MBP peptide p35-47 (MBP 35-47), and acetylated (MBP 1-9).

The effect of the invention complexes on ameliorating disease symptomsin individuals in which EAE has been induced can be measured by survivalrates, and by the progress of the development of symptoms.

Formulation and Administration

If the transmembrane region of the MHC subunit is included, thecomplexes of the invention are conveniently administered after beingincorporated in lipid monolayers or bilayers. Typically liposomes areused for this purpose but any form of lipid membrane, such as planarlipid membranes or the cell membrane of a cell (e.g., a red blood cell)may be used. The complexes are also conveniently incorporated intomicelies. The data presented in Example 2, below, shows that MHC-peptidecomplexes comprising dimeric MHC molecules exist primarily asaggregates.

Liposomes can be prepared according to standard methods, as describedbelow. However, if the transmembrane region is deleted, the complex canbe administered in a manner conventionally used for peptide-containingpharmaceuticals.

Administration is systemic and is effected by injection, preferablyintravenous, thus formulations compatible with the injection route ofadministration may be used. Suitable formulations are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985), which is incorporated herein byreference. A variety of pharmaceutical compositions comprising complexesof the present invention and pharmaceutically effective carriers can beprepared. The pharmaceutical compositions are suitable in a variety ofdrug delivery systems. For a brief review of present methods of drugdelivery, See, Langer, Science 249:1527-1533 (1990) which isincorporated herein by reference.

In preparing pharmaceutical compositions of the present invention, it isfrequently desirable to modify the complexes of the present invention toalter their pharmacokinetics and biodistribution. For a generaldiscussion of pharmacokinetics, see, Remington's PharmaceuticalSciences, supra, Chapters 37-39. A number of methods for alteringpharmacokinetics and biodistribution are known to one of ordinary skillin the art (see, e.g., Langer, supra). For instance, methods suitablefor increasing serum half-life of the complexes include treatment toremove carbohydrates which are involved in the elimination of thecomplexes from the bloodstream. Preferably, substantially all of thecarbohydrate moieties are removed by the treatment. Substantially all ofthe carbohydrate moieties are removed if at least about 75%, preferablyabout 90%, and most preferably about 99% of the carbohydrate moietiesare removed. Conjugation to soluble macromolecules, such as proteins,polysaccharides, or synthetic polymers, such as polyethylene glycol, isalso effective. Other methods include protection of the complexes invesicles composed of substances such as proteins, lipids (for example,liposomes), carbohydrates, or synthetic polymers.

Liposomes of the present invention typically contain the MHC-peptidecomplexes positioned on the surface of the liposome in such a mannerthat the complexes are available for interaction with the T cellreceptor. The transmembrane region is usually first incorporated intothe membrane at the time of forming the membrane. The liposomes can beused to target desired drugs (e.g. toxins or chemotherapeutic agents) toparticular autoreactive T cells. Alternatively, the complexes embeddedin the liposome may be used to induce anergy in the targeted cells.

Liposome charge is an important determinant in liposome clearance fromthe blood, with negatively charged liposomes being taken up more rapidlyby the reticuloendothelial system (Juliano, Biochem. Biophys. Res.Commun. 63:651 (1975)) and thus having shorter half-lives in thebloodstream. Liposomes with prolonged circulation half-lives aretypically desirable for therapeutic and diagnostic uses. For instance,liposomes which can be maintained from 8, 12, or up to 24 hours in thebloodstream are particularly preferred.

Typically, the liposomes are prepared with about 5-15 mole percentnegatively charged phospholipids, such as phosphatidylglycerol,phosphatidylserine or phosphatidylinositol. Added negatively chargedphospholipids, such as phosphatidylglycerol, also serve to preventspontaneous liposome aggregating, and thus minimize the risk ofundersized liposomal aggregate formation. Membrane-rigidifying agents,such as sphingomyelin or a saturated neutral phospholipid, at aconcentration of at least about 50 mole percent, and 5-15 mole percentof monosialylganglioside, may provide increased circulation of theliposome preparation in the bloodstream, as generally described in U.S.Pat. No. 4,837,028, incorporated herein by reference.

Additionally, the liposome suspension may include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asetocopherol and water-soluble iron-specific chelators, such asferrioxianine, are preferred.

A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028, all of which areincorporated herein by reference. One method produces multilamellarvesicles of heterogeneous sizes. In this method, the vesicle-forminglipids are dissolved in a suitable organic solvent or solvent system anddried under vacuum or an inert gas to form a thin lipid film. Ifdesired, the film may be redissolved in a suitable solvent, such astertiary butanol, and then lyophilized to form a more homogeneous lipidmixture which is in a more easily hydrated powderlike form. This film iscovered with an aqueous solution of the targeted drug and the targetingcomponent and allowed to hydrate, typically over a 15-60 minute periodwith agitation. The size distribution of the resulting multilamellarvesicles can be shifted toward smaller sizes by hydrating the lipidsunder more vigorous agitation conditions or by adding solubilizingdetergents such as deoxycholate.

The hydration medium contains the targeted drug at a concentration whichis desired in the interior volume of the liposomes in the final liposomesuspension. Typically the drug solution contains between 10-100 mg/ml ofthe complexes in a buffered saline solution.

Following liposome preparation, the liposomes may be sized to achieve adesired size range and relatively narrow distribution of liposome sizes.One preferred size range is about 0.2-0.4 microns, which allows theliposome suspension to be sterilized by filtration through aconventional filter, typically a 0.22 micron filter. The filtersterilization method can be carried out on a high through-put basis ifthe liposomes have been sized down to about 0.2-0.4 microns.

Several techniques are available for sizing liposome to a desired size.One sizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize discrimination.

Extrusion of liposome through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size.

Even under the most efficient encapsulation methods, the initial sizedliposome suspension may contain up to 50% or more complex in a free(nonencapsulated) form.

Several methods are available for removing non-entrapped compound from aliposome suspension. In one method, the liposomes in the suspension arepelleted by high-speed centrifugation leaving free compound and verysmall liposomes in the supernatant. Another method involvesconcentrating the suspension by ultrafiltration, then resuspending theconcentrated liposomes in a replacement medium. Alternatively, gelfiltration can be used to separate large liposome particles from solutemolecules.

Following the above treatment, the liposome suspension is brought to adesired concentration for use in intravenous administration. This mayinvolve resuspending the liposomes in a suitable volume of injectionmedium, where the liposomes have been concentrated, for example bycentrifugation or ultrafiltration, or concentrating the suspension,where the drug removal step has increased total suspension volume. Thesuspension is then sterilized by filtration as described above. Theliposomes comprising the MHC-peptide complex may be administeredparenterally or locally in a dose which varies according to, e.g., themanner of administration, the drug being delivered, the particulardisease being treated, etc.

Micelles are commonly used in the art to increase solubility ofmolecules having nonpolar regions. One of skill will thus recognize thatmicelles are useful in compositions of the present invention. Micellescomprising the complexes of the invention are prepared according tomethods well known in the art (see, e.g., Remington's PharmaceuticalSciences, supra, Chap. 20). Micelles comprising the complexes of thepresent invention are typically prepared using standard surfactants ordetergents.

Micelles are formed by surfactants (molecules that contain a hydrophobicportion and one or more ionic or otherwise strongly hydrophilic groups)in aqueous solution. As the concentration of a solid surfactantincreases, its monolayers adsorbed at the air/water or glass/waterinterfaces become so tightly packed that further occupancy requiresexcessive compression of the surfactant molecules already in the twomonolayers. Further increments in the amount of dissolved surfactantbeyond that concentration cause amounts equivalent to the new moleculesto aggregate into micelies. This process begins at a characteristicconcentration called "critical micelle concentration".

The shape of micelles formed in dilute surfactant solutions isapproximately spherical. The polar head groups of the surfactantmolecules are arranged in an outer spherical shell whereas theirhydrocarbon chains are oriented toward the center, forming a sphericalcore for the micelle. The hydrocarbon chains are randomly coiled andentangled and the micellar interior has a nonpolar, liquid-likecharacter. In the micelles of polyoxyethylated nonionic detergents, thepolyoxyethlene moieties are oriented outward and permeated by water.This arrangement is energetically favorable since the hydrophilic headgroups are in contact with water and the hydrocarbon moieties areremoved from the aqueous medium and partly shielded from contact withwater by the polar head groups. The hydrocarbon tails of the surfactantmolecules, located in the interior of the micelle, interact with oneanother by weak van der Waals forces.

The size of a micelle or its aggregation number is governed largely bygeometric factors. The radius of the hydrocarbon core cannot exceed thelength of the extended hydrocarbon chain of the surfactant molecule.Therefore, increasing the chain length or ascending homologous seriesincreases the aggregation number of spherical micelies. For surfactantswhose hydrocarbon portion is a single normal alkyl chain, the maximumaggregation numbers consistent with spherical shape are approximately27, 39, 54, 72, and 92 for C₈, C₁₀, C₁₂, C₁₄ and C₁₆, respectively. Ifthe surfactant concentration is increased beyond a few percent and ifelectrolytes are added (in the case of ionic surfactants) or thetemperature is raised (in the case of nonionic surfactants), themicelles increase in size. Under these conditions, the micelles are toolarge to remain spherical and become ellipsoidal, cylindrical or finallylamellar in shape.

Common surfactants well known to one of skill in the art can be used inthe micelles of the present invention. Suitable surfactants includesodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethyleneglycol monododecyl ether, octoxynol 9 and PLURONIC F-127.sup.•(Wyandotte Chemicals Corp.). Preferred surfactants are nonionicpolyoxyethylene and polyoxypropylene detergents compatible with IVinjection such as, TWEEN-80.sup.•, PLURONIC F-68.sup.•,n-octyl-β-D-glucopyranoside, and the like. In addition, phospholipids,such as those described for use in the production of liposomes, may alsobe used for micelle formation.

Since the MHC subunits of the present invention comprise a lipophilictransmembrane region and a relatively hydrophilic extracellular domain,mixed micelles are formed in the presence of common surfactants orphospholipids and the subunits. The mixed micelles of the presentinvention may comprise any combination of the subunits, phospholipidsand/or surfactants. Thus, the micelles may comprise subunits anddetergent, subunits in combination with both phospholipids anddetergent, or subunits and phospholipid.

For pharmaceutical compositions which comprise the complexes of thepresent invention, the dose will vary according to, e.g., the particularcomplex, the manner of administration, the particular disease beingtreated and its severity, the overall health and condition of thepatient, and the judgment of the prescribing physician. Dosage levelsfor murine subjects are generally between about 10 μg and about 500 μg.A total dose of between about 50 μg and about 300 μg, is preferred. Forinstance, in treatments provided over the course of a disease, three 25μg or 100 μg doses are effective. Total dosages range between about 0.5and about 25 mg/kg, preferably about 3 to about 15 mg/kg.

The pharmaceutical compositions are intended for parenteral, topical,oral or local administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment. The pharmaceuticalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include powder, tablets, pills,and capsules.

Preferably, the pharmaceutical compositions are administeredintravenously. Thus, this invention provides compositions forintravenous administration which comprise a solution of the complexdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, and the like. For instance, phosphatebuffered saline (PBS) is particularly suitable for administration ofsoluble complexes of the present invention. A preferred formulation isPBS containing 0.02% TWEEN-80. These compositions may be sterilized byconventional, well-known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of the complex can vary widely, i.e., from less thanabout 0.05%, usually at or at least about 1% to as much as 10 to 30% byweight and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.Preferred concentrations for intravenous administration are about 0.02%to about 0.1% or more in PBS.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient.

For aerosol administration, the complexes are preferably supplied infinely divided form along with a surfactant and propellant. Thesurfactant must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olesteric and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride such as, for example, ethylene glycol, glycerol,erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derivedfrom sorbitol, and the polyoxyethylene and polyoxypropylene derivativesof these esters. Mixed esters, such as mixed or natural glycerides maybe employed. The surfactant may constitute 0.1%"20% by weight of thecomposition, preferably 0.25-5%. The balance of the composition isordinarily propellant. Liquefied propellants are typically gases atambient conditions, and are condensed under pressure. Among suitableliquefied propellants are the lower alkanes containing up to 5 carbons,such as butane and propane; and preferably fluorinated orfluorochlorinated alkanes. Mixtures of the above may also be employed.In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedcompounds and surfactant. The ingredients are thus maintained at anelevated pressure until released by action of the valve.

The compositions containing the complexes can be administered fortherapeutic, prophylactic, or diagnostic applications. In therapeuticapplications, compositions are administered to a patient alreadysuffering from a disease, as described above, in an amount sufficient tocure or at least partially arrest the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as"therapeutically effective dose." Amounts effective for this use willdepend on the severity of the disease and the weight and general stateof the patient. As discussed above, this will typically be between about0.5 mg/kg and about 25 mg/kg, preferably about 3 to about 15 mg/kg.

In prophylactic applications, compositions containing the complexes ofthe invention are administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be a"prophylactically effective dose." In this use, the precise amountsagain depend on the patient's state of health and weight. The doses willgenerally be in the ranges set forth above.

In diagnostic applications, compositions containing the appropriatelycomplexes or a cocktail thereof are administered to a patient suspectedof having an autoimmune disease state to determine the presence ofautoreactive T cells associated with the disease. Alternatively, theefficacy of a particular treatment can be monitored. An amountsufficient to accomplish this is defined to be a "diagnosticallyeffective dose." In this use, the precise amounts will depend upon thepatient's state of health and the like, but generally range from 0.01 to1000 mg per dose, especially about 10 to about 100 mg per patient.

Kits can also be supplied for therapeutic or diagnostic uses. Thus, thesubject composition of the present invention may be provided, usually ina lyophilized form in a container. The complexes, which may beconjugated to a label or toxin, or unconjugated, are included in thekits with buffers, such as Tris, phosphate, carbonate, etc.,stabilizers, biocides, inert proteins, e.g., serum albumin, or the like,and a set of instructions for use. Generally, these materials will bepresent in less than about 5% wt. based on the amount of complex andusually present in total amount of at least about 0.001% wt. based againon the protein concentration. Frequently, it will be desirable toinclude an inert extender or excipient to dilute the active ingredients,where the excipient may be present in from about 1 to 99% wt. of thetotal composition. Where an antibody capable of binding to the complexis employed in an assay, this will usually be present in a separatevial. The antibody is typically conjugated to a label and formulatedaccording to techniques well known in the art.

The following example illustrates, but does not limit, the invention.

EXAMPLE 1 Down-Regulation of T cells In Vitro With A Complex of MouseI-A^(k) and rat MBP Peptide

The efficacy of Class II MHC-peptide complexes in induction ofnonresponsiveness or anergy in T cell clones directed against epitopesof myelin basic protein (MBP), known to induce experimental allergicencephalomyelitis in mice, a disease model which mimics human multiplesclerosis, is shown below.

T cell clones AJ1.2 and 4R3.4 prepared by immunization of mice againstrat MBP peptide (1-11) and characterized for antigen specificity wereobtained from Dr. Pat Jones of Stanford University.

The I-A^(k) complex with rat NSF peptide was formed utilizing purifiedmouse I-A^(k) and synthetic rat MBP peptide (1-13), the sequence forwhich is known (Zamvil et al. (1986), Nature 324:258260), and which is:

    Ac-ASQKRPSQRHGSK

Mouse I-A^(k) was purified by a modified method based upon Turkewitz etal., supra. Basically, a soluble membrane extract of cells containingI-A^(k) was prepared using NP-40. I-A^(k) from the extract was purifiedby affinity chromatography, using a column containing 10-2.16antibodies, which had been purified by affinity chromatography onProtein-A, and which were coupled to CNBr activated Sepharose 4b. Thepreparation schemes for the NP-40 soluble membrane extract, for thepurification of 10-2.16 Mab and its coupling to CnBr activated Sepharose4B, and for the purification of I-A^(k), are shown in FIGS. 12, 13a and14, respectively. FIG. 13b is a copy of a polyacrylamide gel showing thePurity of the purified 10-2.16 antibody. The purity of I-A^(k), asmonitored by polyacrylamide gel analysis, is shown in FIG. 15.

In order to form the complex of I-A^(k) and rat MBP peptide, ten ug ofaffinity-purified I-A^(k) in PBS containing 30 mM octyl glucoside and50-fold molar excess of HPLC-purified MBP peptide were mixed in a totalvolume of 125 ul. Samples were incubated at 37° C. for 16 hours withconstant shaking and were either separated from peptide by G-24 Sephadexdesalting for liposome preparation or, for cell studies, were dialyzedagainst PBS followed by RPMI media for 36 hours at 4° C.

The introduction of the I-A^(K) -MBP peptide complex into liposomes wasas follows. A lipid solution consisting ofcholesterol:dipalmitoylphosphatidyl choline(DPPC):dipalmitoylphosphatidyl ethanolamine-fluorescein (DPPEF) at amolar ration of 25:75:2 was prepared in chloroform containing 30 mMoctyl glucoside (OG). Lipid was dried under vacuum and preformed I-A^(k)-peptide complex in PBS containing 17 mM OG was mixed with dried lipidat a ration of 5:1 (w/w). The mixture was vortexed for 2-3 minutes,cooled to 4° C., and finally dialyzed against PBS followed by RPMI mediafor 36 at 4° C. In experiments using (125-I)-labeled I-A^(k), nofluoresceinated lipid was included in the lipid mixture, and theincorporation of I-A^(k) into liposomes was measured by scintigraphy.

Planar lipid membranes were prepared on sterile 12 mm glass coverslipsusing 50-100 ul of liposomes containing affinity-purified I-A^(k) aloneor purified I-A^(k) +MBP(1-13) by the method of Watts et al. (1985),Proc. Natl. Acad. Sci. USA 82:5480-5484, which is incorporated herein byreference. The presence of I-A^(k) in planar membranes was confirmed byfluorescence microscopy after staining with fluorescent anti-I-A^(k)anti-body. No fluorescence above background was noted upon staining withfluorescent anti-I-A^(d).

AJ1.2 and 4R3.4 cells obtained six to eight days after MBP peptidestimulation were washed twice, and the 4×10⁵ cells were added to planarmembranes. The plates were incubated for 48-72 hours in 5% CO₂ at 37° C.and then examined visually for formation of colonies.

The effects of detergent-solubilized Class II molecules were examined byculturing 1×10⁵ AJ1.2 or 4R3.4 cells with 50-100 ul of purified I-A^(k)alone, purified I-A^(k) plus MBP(1-13) and medium alone for five hoursat 37° C. in 5% CO₂. Following this incubation the cells were diluted to900 ul and tested for their ability to respond to antigen-presentingcells (APC) and antigen [MBP(1-13)] in a proliferation assay. Uptake of3-(4,5-dimethyl-thiazol-2-7')-2,5 diphenyltetrazolium bromide (MTT) wasused as an indication of cell proliferation. Although DNA synthesis,usually monitored by ³ H-thymidine uptake, and the activity ofmitochondria, measured by MTT uptake, are different cellular functions,it has been demonstrated that these two activities, monitored three daysafter initiation of stimulation of spleen cell cultures, tracked eachother very well (Molecular Device Application Bulletin Number 011-A,Feb. 9, 1988).

Data are presented as % suppression of proliferation of cells incubatedwith Class I+Ag compared to cells cultured with medium alone and werecalculated by using the formula: ##EQU1##

Since proliferation of cells cultured in the presence of Class II MHCalone was generally equal to cells cultured with medium alone in moststudies, this latter number was used in obtaining % suppression. TheStandard Deviation of triplicate wells was <10% in the majority ofexperiments.

Initially two qualitative studies were performed to determine whetherpretreatment with I-A^(K) +MBP(1-13) will alter the binding of T cellclones to planar membranes prepared from liposomes containing I-A^(k)+MBP(1-13). AJ1.2 cells were used for these studies because they formedcharacteristic colonies on planar membranes in the presence of MBP(1-13)alone, i.e., without antigen presenting cells (APC). Preincubation ofAJ1.2 cells with I-A^(k) +MBP(1-13) for five hours inhibited the numberof colonies formed on planar membranes compared to cells incubated withI-A^(k) or medium alone. In the second experiment, AJ1.2 cells wereincubated with liposomes containing I-A^(k) +MBP(1-13) or with I-A^(k)alone for five hours and then added to planar membranes prepared asdescribed above. As noted previously with detergent-solubilized I-A^(k)MBP(1-13), culturing of cells with liposome containing I-A^(k)+MBP(1-13) reduced the number of colonies in comparison to cellsincubated with liposomes containing I-A^(k) alone. Although coloniescould not be counted accurately, clear differences in their number wereevident.

Because these studies did not allow quantitation of the effects ofI-A^(k) +MBP(1-13) on the function of T cell clones, we examined theeffects of preincubation with this complex on the proliferation of 4R3.4or AJ1.2 cells in the presence of APCs and MBP(1-13). Therefore, 4R3.4or AJ1.2 cells were preincubated with 50-100 ul of I-A^(k) +MBP(1-13),I-A^(k), or medium alone for five hours at 37° C. The cells were thendiluted to an appropriate concentration and added to APC. Antigen[MBP(1-13)] was added to a final concentration ranging from 13.3 um to53.2 um.

APC used in the study were prepared from spleens of female A/J mice.Briefly, spleens were removed and single cell suspensions were preparedby gentle teasing between the frosted ends of sterile microscope slides.Red cells were lysed by hypotonic shock. The remaining cells were washedtwice with RPMI containing antibiotics and incubated with 10micrograms/ml mitomycin-C for 1 hour at 37° C. Following thisincubation, spleen cells were washed five times with RPMI containingantibiotics, counted, and used as APC's.

Following a 72-hour incubation period of the cells with APC andMBP(1-13), the extent of proliferation was quantitated using MTT uptake.The results of eight such studies are summarized in FIG. 16. In studies1, 3, and 4, 4R3.4 cells were incubated as above. In study 2, 4R3.4cells were preincubated with liposomes containing I-A^(k) +MBP(1-13) orI-A^(k) alone. T cells were then separated from unbound liposomes bycentrifugation through a 10% Ficoll solution, washed, and used inproliferation assays. Studies 5 through 8 were carried out with theclone AJ1.2. Cells incubated with I-A^(k) alone proliferated to the sameextent as cells cultured in medium. T cell clones preincubated in thismanner did not proliferate in the absence of APC.

The data presented above demonstrate that the complex of Class IIMHC+MBP(1-13) induces dramatic nonresponsiveness in T cell clonesspecific for MBP(1-11). In addition, the data show that this complex wasimmunologically reactive with, and hence bound to the MBP-stimulated Tcell clones.

Preparation and stability of human HLA-DR2-MBP complexes

The optimum pH for maximum binding of human MBP(83102) peptide toHLA-DR2 was determined to be pH 7. Affinity-purified DR2 from homozygouslymphoblastoid cells was incubated with 10 fold molar excess ofradioiodinated MBP(83-102) peptide at 37° C. for 48 hours at various pH.The unbound peptide was removed by dialysis and the amount of boundpeptide was calculated from the silica gel TLC assay. Samples were alsoanalyzed on reduced and non-reduced polyacrylamide gel. Bands were cutout, counted and the amount of bound peptide was calculated from thespecific activity.

The stability of human DR2-MBP(83-102) complex at 4° C. was alsoinvestigated. Complexes of DR2 and 125I-MBP(83-102) were prepared asdescribed above and stored at 4° C. Every week an aliquot of 1 μl wasapplied on a silica gel TLC plate in triplicate. Plates were run,developed and the percent dissociation was calculated. Over a period of42 days, there was no significant dissociation of this complex.

EXAMPLE 2 EBV Transformation of B Cells from an Individual With anAutoimmune Dysfunction

Peripheral blood mononuclear cells (PBMNC) from an individual with anautoimmune dysfunction are isolated by diluting whole blood or buffy 1:1with sterile phosphate buffered saline (PBS), pH 7.2, layering thesuspension on Ficoll-Hypaque, and centrifuging 20 minutes at 1800-2000RPM in a table top centrifuge. PBMNC present in a band at the interfaceof the Ficoll-Hypaque and PBS-plasma are harvested with a pipette andwashed twice with PBS. Cells are resuspended at 5×10⁶ cells/ml in RPMI1640 containing 10% fetal serum (FBS), plated in a polystyrene flask andincubated for 1 hour at 37° C. to remove monocytes. Non-adherent cellsare collected, pelleted by centrifugation and resuspended at 10×10⁶cells/ml in Ca⁺⁺ --Mg⁺⁺ free Dulbecco's PBS containing 15% FBS. AET-SRBC(2% v/v) is mixed 1:1 with PBMC, the mixture is centrifuged for 20 min.at 100×g, and then incubated on ice for 1 hour. The pellet is gentlyresuspended, and the suspension centrifuged through Ficoll-Hypaque asdescribed earlier. The band which contains B cells and remainingmonocytes is harvested.

Transformation of B cells is with B95-8 cell line (Walls and Crawford inLymphocytes: A Practical Approach (G. G. B. Klaus ed., IRL Press)). TheB95-8 cells are diluted 1:3 in medium, and cultured for 5 days at 37° C.The supernatant is harvested, centrifuged at 250×g for 15 minutes, andfiltered through a 0.45 micron millipore filter. The EBV is thenconcentrated by centrifugation at 10,000 rpm for 2 hours at 4° C., andthe pellet containing the virus is suspended in RPMI 1640 containing 10%FBS, at 1% of the original volume.

In order to transform B cells, the virus stock is diluted 1:9 withculture medium containing 2×10⁶ cells. After the virus is absorbed tothe cells for 1-2 hours at 4° C. the cells are centrifuged at 250×g. Theresulting cell pellet is suspended at approximately 0.7×10⁶ to 7.0×10⁶cells/ml in RPMI 1640 containing 10% FBS. Transformed cells are clonedusing standard methods.

The transformed B cells made by the procedure are suitable for theisolation of human MHC glycoproteins.

EXAMPLE 3 Induction of EAE in Mice

Adoptive transfer of T cell clones AJ1.2 and 4R3.4, as well asimmunization of mice with MBP(1-13) causes mice to develop EAE.

EAE was induced with the peptide using the method for induction of EAEin mice with intact MBP. Briefly, MBP(1-13) was dissolved in PBS andmixed vigorously with complete Freund's adjuvant so as to form a thickemulsion. Female A/J mice were injected with 100 micrograms of thismixture at four sites on the flank. Twenty-four and 72 hours later, 400ng of pertussis toxin was injected intravenously. Mice are observeddaily by two individuals for the development of EAE and mortality. Theresults in FIG. 17 show the development of EAE in mice resulting fromimmunization with MBP(1-13).

For the adoptive transfer of EAE, T cell clone 4R3.4, obtained fromB10A(4R) strain of mice following immunization with MBP(1-11) was used.B10.A(4R) mice were given 350 rad of whole body radiation and theninjected with 400 ng pertussis toxin intravenously. Two to three hourslater 10×10⁶ 4R3.4 cells, stimulated with MBP(1-13) three dayspreviously, were injected intravenously. These animals were observedtwice daily for signs of EAE and mortality. The results of this studyare summarized in FIG. 18.

EXAMPLE 4 Down-Regulation of EAE by I-A^(s) -MBP(91-103) Complex

This example demonstrates that in vivo therapy with complexes of thepresent invention results in prevention of passively induced EAE. Inaddition, the therapy significantly lowered mortality and morbidity intreated animals.

In order to demonstrate that treatment with I-A^(s) /MBP(91-103) complexwill prevent the development of EAE following T cell activation, SJLmice were injected with MBP(91-103) reactive T cell blasts in vivo.Briefly, SJL mice 10-12 weeks of-age were immunized with 400 μg ofMBP(91-103) (Ac-FFKNIVTPRPPP-amide, >95% purity) in complete Freund'sadjuvant on the dorsum. After 10-12 days, regional draining lymph nodecells were harvested and cultured in 24 well plates (Falcon) at aconcentration 6×10⁶ cells/well in a 1.5 mls of RPMI 1640 mediacontaining 10% fetal bovine serum, 1% penicillin/strepmycin and 50 μg/mlof MBP. Following a 4 day in vitro stimulation, MBP(91-103) reactive Tcell blasts were harvested via ficoll-hypaque gradient (Hypaque 1077,Sigma, Mo.) and washed twice in PBS according to standard techniques.Approximately 1.3-1.5×10⁷ cells were injected into each mouse.

Mice that received encephalitogenic MBP(91-103) reactive T cells thenreceived either 100 μg of soluble I-A^(s) /MBP(91-103) complexes in 100μl PBS, 100 μg of I-A^(s) /MBP(1-14) (a peptide that is notencephalitogenic in SJL mice) complexes in 100 μl PBS or PBS alone ondays 0, 3, and 7 (total dose 300 μg). Animals were observed daily andgraded for clinical signs of EAE: grade 1, loss of tail tone; grade 2,hind leg weakness; grade 3, hind leg paralysis; grade 4, moribund; grade5, death. In accordance with the regulations of the animals carecommittee, mice that could not feed themselves were sacrificed.

Only one of the seven mice that received I-A^(s) /MBP(91-103) complexdeveloped clinical EAE on day 16 (FIG. 19). In contrast, all fouranimals that received the I-A^(s) /MBP1-14, and six of seven animalsthat received PBS developed paralysis. In the former group, the meanonset of disease was on days 9.7 and in the latter group it was 9.0 withmean severity of 2.3 and 2.5 respectively.

The inability of other auto-antigens presented by the I-A^(s) allele toinhibit disease induction was also demonstrated. SJL mice were immunizedwith the peptide 139-151 of proteolipoprotein (PLP) in complete Freund'sadjuvant to induce EAE. SJL mice were immunized with the peptidedissolved in PBS and mixed with complete Freund's adjuvant containing 4mg/ml Myobacterium tuberculosis H37Ra in a 1:1 ratio. Animals wereinjected with 152 μg of peptide, a dose found to induce EAE in 100% ofthe animals, subcutaneously in both abdominal flanks. On the same day,and 48 hours later, all animals were given 400 μg of pertussis toxinintravenously. Mice were treated with PBS, 15 μg of I-A^(s) alone or 15μg of I-A^(s) plus PLP(139-151) on days 1, 4, and 7 after immunizationas described above.

As shown in Table 1, animals that received the appropriate I-A^(s)/PLP(139-151) peptide complex were protected from the severe fulminantparalytic disease induced by the immunization with peptide in adjuvant.The was no mortality in the I-A^(s) /PLP peptide treated group. Althoughall six animals did develop paralysis, the mean severity of animals thatwere paralyzed was 2.2 and the mean day of onset was 10.6. In contrast,all six animals that received I-A^(s) complex alone, died with a meanday of onset of 8.2. Five animals died by day 11 and one animal died onday 21. Animals that received saline or no treatment had a mortality of87% and the average day of onset was 9.2 (p<0.0001 I-A^(s) /PLP(139-151)

                  TABLE 1                                                         ______________________________________                                                                             Day of                                   Treatment No Animals Mean            Onset                                    Received  Paralyzed  Severity Mortality                                                                            Paralysis                                ______________________________________                                        None or   15 of 15   4.7       87%   8                                        Saline                                                                        I-A.sup.s complex                                                                       6 of 6     5        100%   7                                        alone                                                                         I-A.sup.s PLP(139-                                                                      6 of 6     2.2      0      10                                       151)                                                                          ______________________________________                                    

Our observations indicate that in vivo therapy with I-A^(s) /MBP(91-103)complexes (300 μg) results in the prevention of passively induced EAE.In addition, therapy with 45 μg of I-A^(s) /PLP(139-151) significantlylowered the mortality and morbidity in animals that received thistherapy.

The complexes of the invention were also tested for the ability toprevent relapse of EAE in mice. In these experiments, SJL mouse received1.2×10⁷ p91-103 reactive T cells ip on day zero. Initial paralytic signsdeveloped between 8-12 days and all animals recovered by at least 2clinical grades by day 22. The mice received 50 μgm of MHC complex (withthe cognate peptide and without), or PBS iv on days 27, 37, and 47. Inexperiment 1 mice were observed for 102 days and in Experiment 2, theywere observed for 112 days. The results indicated that the complexeswith the cognate peptide were significantly more effective than controlsin preventing relapses.

EXAMPLE 5 Down-regulation of RA by MHCII-HSP (180-188) Complexes

Lewis rats develop a form of arthritis called adjuvant arthritis inresponse to subcutaneous injections of Mycrobacterium tuberculosisemulsified in incomplete Freund's adjuvant. This model of arthritisfulfills many of the criteria necessary for evaluating efficacy of drugsbeing developed for the treatment of rheumatoid arthritis. The pathologyof the tissue and the infiltration of monocytic and lymphocytic cellsindicate a strong T cell mediated response. The experiments describedhere use a technique that anergizes peptide-specific-T cells thatrecognize and bind the peptide-MHC Class II complex. The studies involvein vivo treatment with the soluble MHC Class II-peptide complex soonafter the induction of the disease. The results show significantly lessbone degeneration in the MHC Class II-peptide treated rats compared tothe group that received saline treatment.

Lewis rat MHC Class II RT1B and RT1D molecules were affinity purifiedfrom NP-40 extract of splenocyte membranes on OX-6 and OX-17 monoclonalantibodies coupled to sepharose 4B columns. The relative yield of RT1Bto RT1D was 1:2. MHC Class II molecules were loaded with the peptide bymixing 56 μg of RT1B and 113 μg of RT1D molecules with 50-fold molarexcess of the heat shock protein (HSP) peptide, p(180-188), at 37° C.for 48 h in a total volume of 1 ml phosphate buffer pH 7.5 containing 1%octylglucoside. The unbound peptide was removed by extensive dialysis ofthe sample against PBS buffer at 4° C. The final complex concentrationwas 170 μg/ml and was free of endotoxin as tested by Limulus amebocytelysate procedure as described by Whittaker Bioproduct, Inc.

Six male Lewis rats (age 77 days) were injected in both hind foot padswith 1 mg of Mycobacterium tuberculosis in incomplete Freund's adjuvantto induce arthritis. Three of the rats were treated with the MHC ClassII+ plus HSP180-188 complex intravenously on days 1, 4 and 7 after theinduction of the disease. The other three rats were given saline asabove. Arthritic index was determined by the gross appearance and by thefollowing criteria: 0) no change; 1) slight change in the joints of thedigits; 2) slight to moderate edema of the paw or swelling of more thantwo digits; 3) swelling of the paw with slight scabbiness, moderatecuring of toes and nails; 4) severe swelling of the paw, markedscabbiness, and prominent curing of toes and nails.

The rats treated with MHC Class II-peptide or saline had swelling ofmost of their feet by day 20, with an arthritic index of 4. It is mostlikely that the differences in the swelling seen in some of the rats'feet was a reflection of the amount of MT injected. However, whenradiographs of the feet were taken on day 35 after the induction of thedisease, there was a significant difference between the MHC ClassII-peptide treated and the saline treated groups.

In a second experiment, 18 animals were treated over 5 weeks during thecourse of disease progression. As illustrated in Table 2, animalstreated with MHC-peptide of the present invention had a greatly reducedarthritic index and significantly reduced joint swelling compared withthe saline treated group. Animals received 25 μg of MHC-peptide on days4, 8, and 12.

                  TABLE 2                                                         ______________________________________                                        Reduction of inflammation and severity of disease                                                   Thickness Arthritic                                     Treatment  # of Rats  (mm ± SD)                                                                            Index                                         ______________________________________                                        Saline     4          10.5 ± 0.6                                                                           4 ± 0                                      MHC alone  4           9.0 ± 0.7*                                                                          3.25 ± 0.5                                 MHC +      5          7.88 ± 1.4*                                                                           2.6 ± 0.55                                HSP(180-188)                                                                  Normal     5          5.55 ± 0.36                                                                          00                                            ______________________________________                                         *Statistically significant compared to saline treatment (p < 0.05 by          student's ttest).                                                        

On day 35, tarsal joints of all animals were measured using verniercalipers. Measurements represent the sum of the thickness of both hindfeet.

EXAMPLE 6 Increasing Serum Half-life of the Complexes

This example presents data showing that various modifications of thecomplexes lead to increased serum half-life.

The protocol for these studies was generally as follows:Affinity-purified, soluble MHC molecules were labeled with ¹²⁵ I by theiodobeads method (Pierce Chemical Co., Rockford, Ill.). Excess ¹²⁵ I wasremoved by dialysis against PBS containing 0.1% neutral detergent. Thequality of the labeled protein was assessed by thin layerchromatography, cellulose acetate electrophoresis, and polyacrylamidegel electrophoresis. The MHC glycoprotein was administered by tail veininjection to mice subjected to Lugol's solution in the drinking water atleast one day before injection. Blood samples were obtained at differenttime points. The animals were then sacrificed to obtain organs ofinterest. Radioactivity in the blood and organ samples was detected in agamma well counter according to standard techniques.

A. Effect of asialoletuin on serum half-life

IA^(k) was labeled and administered to mice as described above. The micewere divided into three sets: (1) I-A^(k) (10 μg i.v.); (2) I-A^(k) (10μg i.v.) plus asialoletuin (10 mg i.v.) plus asialoletuin (100 mg i.p.);and (3) I-A^(k) (10 μg i.v. ) plus asialoletuin (10 mg i.p.). Blood wasdrawn at different time points and the percent of injected dose retainedin the blood was calculated.

The mean serum half-life for the three sets was as follows:

Set 1-3 min.

Set 2-40 min.

Set 3-35 min.

B. Effect of liposomes on serum half-life

I-A^(s) was labeled as described above. The labeled I-A^(s) moleculeswere captured inside liposomes by standard procedures (see, e.g.,Remington's, supra). Ten mice were injected with 10 μg I-A^(s), asdescribed above, and divided into four sets: (1) I-A^(s) alone; (2)liposomal I-A^(s) ; (3) liposomal I-A^(s) coinjected with blankliposomes; and (4) blank liposomes plus, 10 minutes later, liposomalI-A^(s) coinjected with blank liposomes. Blood samples were obtained atdifferent time points after injection, and the percent of injected doseof I-A^(s) retained in the blood was calculated.

The serum mean half-life of the three sets was as follows:

Set 1-2 min.

Set 2-7 min.

Set 3-10 min.

Set 4-60 min.

C. Effect of periodate/cyanoborohydride treatment on serium half-life

I-A^(k) in a phosphate buffer containing 3 mM taurodeoxycholate at pH7.5 was labeled as described above. The labeled molecules were subjectedto periodate oxidation and cyanoborohydride reduction for 5 or 21 hoursat 4° C., using 20 mM sodium periodate and 40 mM cyanoborohydride (finalconcentrations) in 0.1 M acetate buffer at pH 5.5. The reaction wasquenched by addition of ethylene glycol (final concentration 0.7%). Thetreated I-A^(k) was purified by dialysis and administered (10 μg i.v.)to mice, as described above. Nine mice were divided into three sets: (1)I-A^(k) (untreated); (2) I-A^(k) (5 hr. treatment); and (3) I-A^(k) (21hr. treatment). Blood samples were obtained at different time points andthe percent of injected dose of I-A^(k) retained in the blood wascalculated.

The serum half-life of the three sets was as follows:

Set 1-4 min.

Set 2-7 min.

Set 3-70 min.

EXAMPLE 7

The results presented below demonstrate that MHC-peptide complexes ofthe invention exist primarily as aggregates in the absence of detergent.

An IA^(k) -P complex labeled with ¹²⁵ I was prepared as described above.The complex (1.5 mg/ml) was dialysed extensively against phosphatebuffered saline (PBS) to remove detergent and loaded on a 6 ml b.v.Sephadex-G200 column (fractionation size 5000-600,000). The resultspresented in FIG. 20 show that the aggregated complexes pass through thecolumn with the void volume and thus have a molecular weight greaterthan 600,000.

The same dialyzed IA^(k) complex (1.5 mg/ml) was also centrifuged andthe pellet was counted. To do this, 200 μl of complex (300 μg) wasdiluted in 5 ml PBS and centrifuged in a fixed angle rotor at 100,000×gfor 60 minutes. The results are given in Table 3, below.

                  TABLE 3                                                         ______________________________________                                        DETECTION OF COMPLEX AGGREGATION                                              BY HIGH SPEED SPIN                                                            EXP # Starting cpm                                                                             cpm in pellet                                                                            cpm in sup                                                                            % aggreg.                                 ______________________________________                                        1     514,576    317,717    205,797 60.68                                     2     519,340    321,304    209,108 60.57                                     ______________________________________                                    

The results of the chromatography and centrifugation experiments bothshow that MHC-peptide complexes exist largely in aggregated or micellarform. These results strongly indicate that the single subunit complexesof the present invention are also aggregated or in micellar form, in theabsence of detergent.

EXAMPLE 8

This example demonstrates that administration of soluble MHC classII-AChR α peptide 100-116 complexes alter the function of AChR-reactiveT cells and thereby modulate the course of EAMG, an antibody mediated,but T cell dependent autoimmune disease.

AchR and AchR Peptides

Electroplax tissue from Torpedo californica (Pacific Biomarine) washomogenized and the membrane fraction was detergent solubilized (2%Triton X-100, 100 mM NaCl, 10 mMMOPS, 0.1 mM EDTA, 0.02% NAN₃). AChR wasisolated from the solubilized membranes by affinity chromatography withthe anti-AChR monoclonal antibody mAb 35 and dialized against 1.0%n-octyl β-D-glucopyranoside (OG)/PBS.

Torpedo AChRe subunit peptide 100-116 (YAIVHMTKLLLDYPGKI) wassynthesized by solid-phase 9-fluorenylmethoxycarbonyl (FMOC) procedures,using standard procedures. The peptides were purified by reverse-phaseHPLC, and characterized by HPLC and mass spectroscopy.

Rat MHC II Purification and Peptide Loading

Rat class II RT1.B and RT1.D molecules were detergent solubilized (0.5%NP-40, 10 mM Tris HCl pH 8.3, 1 mM PMSF, 0.02% NAN₃) from homogenizedspleens and purified by affinity chromatography with monoclonalantibodies OX 6 and OX 7. The RT1.B/D mixture was incubated at 37° C.for 24 hours with a 50-fold molar excess of peptide AChRα 100-116,followed by 24 hours of dialysis at 4° C. against 0.1% OG/PBS to removeunbound peptide. Analysis by nitrocellulose filter binding and TLC (B.Nag et al. (1991) J. Immunol. Meth. 142: 105) revealed 70-80% of theRT1.B and 90-100% of the RT1.D bound peptide AChRα 100-116. This complexis stable at 4° C. for at least 8 weeks.

Induction of EAMG and Treatment with Soluble MHC II:AchRα 100-116

Male Lewis rats (8-12 weeks old) were injected in both hind footpadswith Tc AchR emulsified in complete Freund's adjuvant, andintraperitoneally with 600 ng of Pertussis toxin. Sick rats (clinicalstage 1-3) were fed moist chow and teeth were trimmed weekly.

On days 1, 4, and 7 post AChR immunization, individual rats were treatedi.v. with saline, 25 μg MHC II alone (MHC II:0), or 25 μg of MHC IIcomplexed with the T.c. AchRα peptide 100-116 (MHC II:AchRe 100-116). Tcells were purified from popliteal lymph nodes 9 days after AchRimmunization and tested for proliferation to a panel of antigens.

T Cell Proliferation Assay

T cells from the peripheral lymph nodes were isolated by nylon woolchromatography. 2×10⁵ T cells and 3×10⁵ irradiated syngeneic splenocyteswere incubated in 0.2 ml of culture medium (RPMI 1640, 10% FBS, 10 mMHEPES, 5×10⁻⁵ M 2-ME, 100 U/m. penicillin, 100 U/ml streptomycin) withpeptide or whole antigen for 3 days at 37° C., 5% CO₂, pulsed with 1 μCi³ H-thymidine for 18 hours, harvested, and counted.

Timecourse of EAMG in Lewis Rats

Immunization of Lewis rats with T. c. AchR induces anti-AchR antibodies,causing weight loss and progressive muscle weakness with a predictabletimecourse. A drop in weight in the week following immunizationcoincides with an "acute phase" due to infiltration of the neuromuscularjunction by mononuclear cells. A second, sustained weight loss begins atday 33-40, a "chronic phase" accompanied by the clinical symptomsresembling MG in human patients. Weight changes of individual rats areshown to demonstrate the range of disease onset, progression, and timeof death (indicated by intersection with the abscissa).

In untreated rats, progressive EAMG may be classified into three stages.In stage 1, anti-AChR antibodies cause endocytosis of the AchR.Subthreshold levels of AChR at the neuromuscular junction reduce musclecontractions, particularly in the posterior muscles. Weak back and hindleg muscles cause a characteristic hunched posture at rest. Stage 2 ischaracterized by progressive weakness that causes frequent rest periods,with the head unsupported due to weakness in the neck muscles. In stage3, the diaphragm and intercostal muscles are weakened, causing laboredbreathing. Deterioration of the mandible muscles leads to excessivetooth growth.

To test the therapeutic effect of soluble MHC I:AChRα 100-116, rats weretreated after clinical symptoms appeared in the chronic phase.

Antigen-Specific Unresponsiveness Induced by Soluble MHC II:AChRα100-116

In saline treated rats, the T cell response to AChRα peptide 100-116equalled 30% of the response to AChR (α₂ β.sub.γ δ) (FIG. 21),confirming literature reports that 100-116 is a major epitope in the Tcell response to AChR. T cells from the MHC:0 treated rat respond toeach antigen at levels similar to T cells from the saline treated rat.

The T cell response of MHC II:AChRα 100-116 treated rats to whole TcAChR and AChRα 100-116 were respectively 22% and 20% of saline treatedrat T cell proliferation levels (FIG. 21). Proliferation of T cells fromthe MHC II:AChRα 100-116 treated rat to Pertussis toxin was equivalentto T cells from saline and MHC II:0 treated rats, indicating that the Tcell inactivation was antigen specific (FIG. 21).

Therapeutic Effect of Soluble MHC II:AChRα 100-116

Randomized groups of rats with clinical stage 1 EAMG (approximately day42-56 post-inoculation) were injected i.v. at five weekly intervals withsaline, 25 μg MHC II:HSP 180-188 (MHC II bearing an irrelevant heatshock peptide), 25 μg MHC II alone (MHC II:0), or 5 μg AChRα 100-116alone (0:AChRα 100-116). The weight and clinical symptoms weremonitored.

Treatment of rats with clinical stage 1 EAMG with MHC II:AChRα 100-116resulted in a survival frequency of 67% at 140 days post-induction. Incomparison, the maximum survival rate in the control groups was 20%(16.7% saline, 0% Tc AChRα 100-116 alone, 20% MHC II alone, 20% MHCII:HSP 180-188). The time course of EAMG for representative rats in eachtreatment group is presented in Table 4.

The four surviving MHC II:AChRα 100-116 treated rats exhibitedrelatively severe EAMG (maxima of 2.0, 2.5, 2.5, and 3.0), followed byremission approximately three weeks after the first therapeuticinjection.

                  TABLE 4                                                         ______________________________________                                                     DAYS POST EAMG INDUCTION                                         TREATMENT      61    123         224                                          ______________________________________                                        MHC II:AChRα 100-116                                                                   2.5   0.0         0.0                                          MHC II:HSP 180-188                                                                           3.0   3.0         Dead (day 138)                               MHC II:0       3.0   Dead (day 66)                                            0:AChR 100-116 2.5   Dead (day 66)                                            Saline         3.0   Dead (day 82)                                            ______________________________________                                    

Representative rats from each treatment group are shown 61, 123, and 224days after EAMG induction by AChR immunization. The clinical stage ofeach rat is indicated. By day 123, the MHC II:AChRα 100-116 treated ratshows improved mobility and posture, in contrast to the lone survivingrat treated with MHC:HSP 180-188.

These results show that soluble MHC II:AChRα 100-116 complex injected atthe start of EAMG induction significantly reduces the T cell response tothe AChRα peptide 100-116 and to whole AChR. The effect of the solubleMHC II:AChRα 100-116 complex is antigen-specific, as the complex doesnot affect the T cell response to an unrelated antigen, pertussis toxin.Treatment with the soluble MHC II:AChRα 100-116 complex during thechronic phase of EAMG reduces mortality and clinical symptoms of thedisease.

EXAMPLE 9

This example demonstrates the ability of complexes of the invention toinduce anergy in human T cells associated with myasthenia gravis (MG).

The T cells in this experiment were derived form the thymus of youngonset MG patient. They recognize the residues 138-167 AChRα subunit andare restricted by DR4Dw4. In the experiments, 2×10⁴ T cells werepreincubated for varying times with either complex (DR4:p138-167), DR4alone, or peptide alone. Antigen presenting cells (APC's) pulsed withdifferent antigens were then added and proliferation of the T cells wasmeasured using tritiated thymidine as described above. The stimulatingantigens included p138-167 and a recombinantly produced AChRα subunitpolypeptide termed r37-181, which was prepared as described in Beeson etal., EMBO J 9:2101-2106 (1990) and Beeson et al., Biochem. Soc. Trans.17:219-220 (1989), both of which are incorporated herein by reference.

Preincubation of the T cells with complex at 10 μgm/ml overnight lead tononresponsiveness in the T cells when subsequently presented withantigen. Incubation with lower concentrations and for shorter times werenot as effective at inducing anergy.

Viability of the anergized cells was tested with Trypan Blue. T-cellswere incubated with media, or complex at 5 μg/ml, and aliquots withdrawnfor counting at various times. Cells were counted as either living ordead based on trypan blue exclusion in living cells. The results arepresented in FIGS. 22A and 22B. In the media incubated cells, the totalnumber of cells gradually rose while the number of dead cells remainedthe same. In the complex incubated cells, the total number of cellsfell--rapidly during the first 6 hours and more slowly thereafter. Thenumber of dead cells gradually rose so that after several daysincubation, almost 90% of the cells were dead. These results show thatcell death follows the induction of anergy in T cells.

To test the specificity of the interaction of the complexes with the Tcells, T-cells (1×10⁴) were incubated with various forms of the complex.These were--the relevant complex DR4:p138-167, DR4 alone, soluble DR4and "sham" p138-167 in equimolar (proportionally) amounts but not boundtogether and DR4:MBPp1-14 complex. Sham peptide was used as previousexperiments had shown that soluble p138-167 alone had similar effects tothe complex. Sham p138-167 was a sample of p138-167 subjected to thesame preparation as the complex including dialysis, thus controlling forthe effects of any soluble peptide which may be released from thecomplex. After overnight preincubation, irradiated PBL's (2×10⁵) andstimulating antigens were added.

Irrelevant complexes and peptides did not induce anergy. Incubationovernight with media and subsequent stimulation with recα37-181 resultedin a good proliferative response. Preincubation with DR4:p13B-167substantially reduced subsequent proliferation. However, preincubationwith any of the other substances had no effect on proliferation comparedto preincubation with media.

In addition to the effects of preincubating with various complexes, theeffect of adding various antibodies to cell surface markers, or ofadding IL2 0.5%, at the time of preincubation with DR4:p138-167 wasstudied. Addition of an anti-TCR Ab (anti-TAC--786) inhibited thebackground stimulation caused by the complex, inhibited anyantigen-induced response and partially inhibited the response tostimulation with IL2 after preincubation. An anti-DR Ab (L234) had noeffects on the inhibition of antigen induced response induced bypreincubation with complex. Combination of anti-TAC and anti-DR reducedbackground proliferation and antigen-induced proliferation but had noeffect on IL2-induced proliferation. Preincubation of the T-cells withcomplex in the presence of 0.5% IL2 did not diminish the inhibitoryeffects of the complex.

Similarly, experiments designed to identify any non-specific effects ofthe complex on non-DR4, non-AChR T cell lines. Cells (2×10⁴) from linesraised against KLH or Tetanus Toxoid were preincubated overnight withthe complex. They were then stimulated in the with irradiated PBL's(2×10⁵) and antigen. Preincubation with the complex did not have anyeffect on background proliferation response to specific antigen. Thusthe complex does not appear to have any non-specific stimulatory orinhibitory effects on unrelated cell lines.

Finally, FIGS. 23A and 23B show that the molar concentration of complexrequired to induce anergy is much less than that of peptide alone. Ascan be seen form the figures, preincubation of T-cells with p138-167 atconcentrations above 1.7×10⁻⁸ resulted in a decrease in the response torecombinant. Preincubation with lower concentrations caused an increasein background proliferation compared to preincubation with media (farleft). Not until concentrations of 1.7×10⁻⁵ were reached did theproliferative response fall to background levels.

Preincubation of T-cells with DR4:p138-167 at concentrations greaterthan 1.7×10⁻¹¹ inhibited the antigen-induced proliferative response. Atconcentrations above 1.7×10⁻⁸ the antigen-induced proliferation fell tobackground levels. The lowest concentration of the complex used,1.7×10⁻¹¹, resulted in an increase in antigen-induced proliferation, anincrease in background proliferation and an increase in theproliferative response to IL2.

Thus, both complex and p138-167 alone cause some stimulation. At higherconcentrations, both inhibit antigen-specific response. However, theamount of p138-167 required for comparable inhibition (i.e. to levels ofbackground stimulation) was approximately 1000× (molar) that ofDR4:p138-167.

The results described in the Examples, above, demonstrate the ability ofthe complexes of the present invention to treat autoimmune disease invivo. These data in combination with the in vitro data showing inductionof anergy establish the effectiveness of the claimed complexes. Althoughthe invention has been described in some detail in these examples forpurposes of clarity and understanding, it will be apparent that certainchanges and modifications may be practiced within the scope of theappended claims.

What is claimed is:
 1. An MHC Class II-peptide complex capable ofinducing nonresponsiveness in a target T cell, the complex consisting ofa preselected antigenic peptide and an isolated MHC Class II component,the MHC Class II component having an antigen binding pocket to which theantigenic peptide is bound so that the antigenic peptide is recognizedby the target T Cell.
 2. The complex of claim 1, wherein the antigenicpeptide comprises an epitope recognized by a T cell specificallyimmunoreactive with an autoantigen.
 3. The complex of claim 2, whereinthe autoantigen is selected from the group consisting of myelin basicprotein, acetyl choline receptor, and type II collagen.
 4. The complexof claim 1, wherein the antigenic peptide consists of between about 8and about 20 amino acids.
 5. The complex of claim 1, wherein theantigenic peptide comprises residues 83-102 of human myelin basicprotein.
 6. The complex of claim 1, wherein the MHC class II componentis isolated from spleen cells.
 7. The complex of claim 1, wherein theMHC Class II component is DR2.
 8. The complex of claim 1, wherein theantigenic peptide is noncovalently associated with the antigen bindingpocket.
 9. A complex consisting of a preselected antigenic peptide, anisolated MHC Class II component having an antigen binding pocket, and aneffector component, the antigenic peptide being bound to the antigenbinding pocket so that the antigenic peptide is recognized by the targetT cell and the effector being bound to the MHC Class II component or theantigenic peptide.
 10. The complex of claim 9, wherein the effectorcomponent is a toxin.
 11. The complex of claim 9, wherein the effectorcomponent is covalently linked to the MHC Class II component.
 12. Thecomplex of claim 9, wherein the effector component is covalently linkedto the antigenic peptide.
 13. The complex of claim 9, wherein theantigenic peptide consists of between about 8 and about 20 amino acids.14. The complex of claim 9, wherein the antigenic peptide comprises anepitope recognized by a T cell specifically immunoreactive with anautoantigen.
 15. The complex of claim 14, wherein the autoantigen isselected from the group consisting of myelin basic protein, acetylcholine receptor, and type II collagen.
 16. The complex of claim 9,wherein the MHC Class II component is isolated from spleen cells. 17.The complex of claim 9, wherein the peptide is noncovalently associatedwith the antigen binding pocket.
 18. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a complex of claims1 or
 9. 19. A method of treating autoimmune disease in a mammal, themethod comprising administering to the mammal a therapeuticallyeffective dose of the pharmaceutical composition of claim
 18. 20. Themethod of claim 19, wherein the pharmaceutical composition isadministered intravenously.
 21. The method of claim 19, wherein thetherapeutically effective dose is between about 3 mg MHC-peptide complexper kg body weight and about 15 mg MHC-peptide complex per kg bodyweight.
 22. The method of claim 19, wherein the autoimmune disease isrheumatoid arthritis, myasthenia gravis, or multiple sclerosis.